Full-length interferon gamma polypeptide variants

ABSTRACT

The present invention relates to novel full-length interferon gamma (IFNG) polypeptide variants having interferon gamma activity. The full-length interferon gamma polypeptide variants of the invention are obtained by performing selected modifications in the C-terminal part of the molecule. The full-length interferon gamma polypeptide variants of the invention are useful in therapy, in particular for the treatment of interstitial pulmonary diseases, such as idiopathic pulmonary fibrosis.

FIELD OF THE INVENTION

The present invention relates to novel full-length interferon gammapolypeptide variants having interferon gamma (IFNG) activity, methodsfor their preparation, pharmaceutical compositions comprising thevariants and their use in the treatment of diseases, in particular forthe treatment of interstitial pulmonary diseases, such as idiopathicpulmonary fibrosis.

BACKGROUND OF THE INVENTION

Interferon gamma (IFNG) is a cytokine produced by T-lymphocytes andnatural killer cells and exists as a homodimer of two noncovalentlybound polypeptide subunits. The mature form of each monomer comprises143 amino acid residues (shown in SEQ ID NO:1) and the precursor formthereof, including the signal sequence, comprises 166 amino acidresidues (shown in SEQ ID NO:2).

Each subunit has two potential N-glycosylation sites (Aggarwal et al.,Human Cytokines, Blackwell Scientific Publications, 1992) at positions25 and 97. Depending on the degree of glycosylation the molecular weightof IFNG in dimer form is 34-50 kDa (Farrar et al., Ann. Rev. Immunol,1993, 11:571-611).

The primary sequence of wild-type human IFNG (huIFNG) was reported byGray et al. (Nature 298:859-863, 1982), Taya et al. (EMBO J. 1:953-958,1982), Devos et al. (Nucleic Acids Res. 10:2487-2501, 1982) andRinderknecht et al. (J. Biol. Chem. 259:6790-6797, 1984), and in EP77670, EP 89676 and EP 110044.

Experimental 3D structures of huIFNG determined by X-ray crystallographyhave been reported by Ealick et al. Science 252:698-702 (1991) whoreported the C-alpha trace of an IFNG homodimer. Walter et al. Nature376:230-235 (1995) disclosed the structure of an IFNG homodimer incomplex with two molecules of a soluble form of the IFNG receptor. Thecoordinates of this structure, however, have never been made publiclyavailable. Thiel et al. Structure 8:927-936 (2000) showed the structureof an IFNG homodimer in complex with two molecules of a soluble form ofthe IFNG receptor having a third molecule of the receptor in thestructure not making interactions with the IFNG homodimer.

Various naturally-occurring or mutated forms of the IFNG subunitpolypeptides have been reported, including one comprising a Cys-Tyr-CysN-terminal amino acid sequence (positions (−3)-(−1) relative to SEQ IDNO:1), one comprising an N-terminal methionine (position −1 relative toSEQ ID NO:1), and various C-terminally truncated forms comprising127-134 amino acid residues. It is known that 1-15 amino acid residuesmay be deleted from the C-terminus without abolishing IFNG activity ofthe molecule. Furthermore, heterogeneity of the huIFNG C-terminus wasdescribed by Pan et al. (Eur. J. Biochem. 166:145-149, 1987).

Glycosylation variation in huIFNG has been reported by Curling et al.(Biochem. J. 272 :333-337, 1990) and Hooker et al., (J. of Interferonand Cytokine Research, 1998, 18: 287-295).

Polymer-modification of huIFNG was reported by Kita et al. (Drug Des.Deliv. 6 :157-167, 1990), and in EP 236987 and U.S. Pat. No. 5109120.

WO 99/03887 discloses PEGylated variants of polypeptides belonging tothe growth hormone superfamily, wherein a non-essential amino acidresidue located in a specified region of the polypeptide has beenreplaced by a cysteine residue. IFNG is mentioned as one example of amember of the growth hormone super family, but modification thereof isnot discussed in any detail.

WO 01/36001 discloses novel IFNG conjugates comprising a non-polypeptidemoiety attached to an IFNG polypeptide which have been modified byintroduction and/or deletion of attachment sites for suchnon-polypeptide moieties, e.g. PEG and glycosylation sites. Thesemolecules have improved properties, such as improved half-life and/orincreased bioavailablity.

IFNG has been suggested for treatment of interstitial lung diseases(also known as Interstitial Pulmonary Fibrosis (IPF) (Ziesche et al. (N.Engl. J. Med. 341:1264-1269, 1999 and Chest 110:Suppl:25S, 1996) and EP0 795 332) for which purpose IFNG can be used in combination withprednisolone. In addition to IPF, granulomatous diseases (Bolinger etal, Clinical Pharmacy, 1992, 11:834-850), certain mycobacterialinfections (N. Engl. J. Med. 330:1348-1355, 1994), kidney cancer (J.Urol. 152:841-845, 1994), osteopetrosis (N. Engl. J. Med. 332:1594-1599,1995), scleroderma (J. Rheumatol. 23:654-658, 1996), hepatitis B(Hepatogastroenterology 45:2282-2294, 1998), hepatitis C (Int. Hepatol.Communic. 6:264-273, 1997), septic shock (Nature Medicine 3:678-681,1997), and rheumatoid arthritis may be treated with IFNG. Furthermore,IFNG is presently being clinically evaluated for treatment of ovariancancer, liver fibrosis, asthma and lymphoma.

As a pharmaceutical compound huIFNG is used with a certain success,above all, against some viral infections and tumors. huIFNG is usuallyapplicable via parenteral, preferably via subcutaneous, injection.Maximum serum concentrations have been found after seven hours. It hasbeen reported that the half-life in plasma is 30 minutes afterintravenous administration. For this reason efficient treatment withhuIFNG involves frequent injections.

The main adverse effects consist of fever, chills, sweating, headache,myalgia and drowsiness. These effects are associated with injectinghuIFNG and are observed within the first hours after injection. Rareside effects are local pain and erythema, elevation of liver enzymes,reversible granulo- and thrombopenia and cardiotoxicity.

It is known that when IFNG is produced in mammalian cell lines aheterogenous population of IFNG polypeptides is obtained due toC-terminal truncation of the IFNG polypeptide (reviewed in Lundell etal. Pharmac. Ther. 64, 1-21, 1994). Clearly, this constitutes a severeproblem in that valuable polypeptide material is lost and, further, itis necessary to carry out time-consuming and cumbersome purification inorder to obtain a homogenous population of IFNG polypeptides having thedesired length. Most likely, this truncation is effected by endo- and/orexoprotease activity present in the cell. The present inventors havesolved the above-mentioned problem by performing selected modificationsin the C-terminal part of the IFNG polypeptide. By performing suchmodifications C-terminal truncation is avoided and, consequently, thetruncation can be controlled and homogenous populations of full-lengthIFNG polypeptides can be obtained.

Thus, it is an object of the present invention to provide novelfull-length IFNG polypeptides, which are not prone to C-terminaltruncation during production or storage.

BRIEF DISCLOSURE OF THE INVENTION

In a first aspect the present invention relates to a full-lengthinterferon gamma (IFNG) polypeptide variant exhibiting IFNG activity,wherein said variant comprises

-   -   (a) at least one amino acid substitution in a position selected        from the group consisting of S132 and S142; and    -   (b) at least one amino acid substitution in a position selected        from the group consisting of R137, R139 and R140.

In a second aspect the present invention relates to a full-lengthinterferon gamma (IFNG) polypeptide variant exhibiting IFNG activity,wherein said variant comprises an amino acid substitution in positionR137 and an amino acid substitution in position R140.

In further aspects the invention relates to means and methods forpreparing an IFNG polypeptide variant of the invention, includingnucleotide sequences and expression vectors encoding the variant as wellas host cells comprising the vector or nucleotide sequence of theinvention.

In even further aspects the present invention relates to apharmaceutical composition comprising the variant of the invention and apharmaceutically acceptable diluent, carrier or adjuvant; to a variantof the invention for use as a medicament; to use of a variant of theinvention for the manufacture of a medicament for the treatment ofinterstitial pulmonary diseases as well as to a method for treating orpreventing interstitial pulmonary diseases, said method comprisingadministering to a mammal, in particular a human being, in need thereofan effective amount of a variant of the invention.

In still another aspect the present invention relates to a method forproducing a full-length IFNG polypeptide, said method comprising

-   -   i) cultivating a host cell according to the invention under        conditions suitable for production of the IFNG polypeptide, and    -   ii) recovering the IFNG polypeptide.

Other aspects of the invention will become apparent from the belowdisclosure.

BREIF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T]huIFNG purifiedfrom culture media by diafiltration followed by cation exchange,immunoprecipitation and de-glycosylation with PNGase F. See Example 8for further details.

FIG. 2 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P]huIFNGpurified from culture media by diafiltration followed by cationexchange, immmunoprecipitation and de-glycosylation with PNGase F. SeeExample 8 for further details.

FIG. 3 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R139P]huIFNGpurified from culture media by diafiltration followed by cationexchange, immunoprecipitation and de-glycosylation with PNGase F. SeeExample 8 for further details.

FIG. 4 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+S142P]huIFNGpurified from culture media by diafiltration followed by cationexchange, immunoprecipitation and de-glycosylation with PNGase F. SeeExample 8 for further details.

FIG. 5 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+Q143P]huIFNGpurified from culture media by diafiltration followed by cationexchange, immunoprecipitation and de-glycosylation with PNGase F. SeeExample 8 for further details.

FIG. 6 shows a MALDI-TOP mass spectra of[E38N+S40T+S99T+R137P+R139P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 7 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+R137P+R139P+Q143P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 8 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+R137P+R139P+S142P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 9 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+R137P+S142P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 10 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+S132P+R137P+R140P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 11 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+S132P+R140P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

FIG. 12 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R140P]huIFNGpurified from culture media by diafiltration followed by cationexchange, immunoprecipitation and de-glycosylation with PNGase F. SeeExample 8 for further details.

FIG. 13 shows a MALDI-TOF mass spectra of[E38N+S40T+S99T+R137P+R140P]huIFNG purified from culture media bydiafiltration followed by cation exchange, immunoprecipitation andde-glycosylation with PNGase F. See Example 8 for further details.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “conjugate” (or interchangeably “conjugated polypeptide”) isintended to indicate a heterogeneous (in the sense of composite orchimeric) molecule formed by the covalent attachment of one or morepolypeptide variant(s) to one or more non-polypeptide moieties. The termcovalent attachment means that the polypeptide variant and thenon-polypeptide moiety are either directly covalently joined to oneanother, or else are indirectly covalently joined to one another throughan intervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties. Preferably, a conjugated polypeptide variant issoluble at relevant concentrations and conditions, i.e. soluble inphysiological fluids such as blood. Examples of conjugated polypeptidevariants of the invention include glycosylated and/or PEGylatedpolypeptides. The term “non-conjugated polypeptide” may be used aboutthe polypeptide part of the conjugated polypeptide variant.

The term “non-polypeptide moiety” is intended to indicate a moleculethat is capable of conjugating to an attachment group of the IFNGvariant. Examples of such molecules include polymer molecules,lipophilic compounds, sugar moieties or organic derivatizing agents.Prefererred examples include polymer molecules, such as PEG, and sugarmoieties. It will be understood that the non-polypeptide moiety islinked to the variant through an attachment group of the variant. Exceptwhere the number of non-polypeptide moieties, such as polymermolecule(s), attached to the IFNG variant is expressly indicated everyreference to “a non-polypeptide moiety” attached to the IFNG variant orotherwise used in the present invention shall be a reference to one ormore non-polypeptide moieties attached to the IFNG variant.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue. The term “polymer” may be used interchangeably withthe term “polymer molecule”.

The term “sugar moiety” is intended to indicate a carbohydrate moleculeattached by in vivo or in vitro glycosylation, such as N— orO-glycosylation.

An “N-glycosylation site” has the sequence N—X—S/T/C″, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine. The terms “N-glycosylation site” and “in vivoN-glycosylation site” are used interchangeably herein. An“O-glycosylation site” is the OH-group of a serine or threonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group capable of coupling to the relevant non-polypeptide moietysuch as a polymer molecule or a sugar moiety. Useful attachment groupsand their matching non-polypeptide moieties are apparent from the tablebelow. Examples of Conjugation Attachment non-polypeptidemethod/activated group Amino acid moiety PEG Reference —NH₂ N-terminal,Lys Polymer, e.g. PEG mPEG-SPA Shearwater Inc. Delgado et al, Tresylatedcritical reviews in Therapeutic mPEG Drug Carrier Systems 9(3, 4):249-304 (1992) —COOH C-term, Asp, Polymer, e.g. PEG mPEG-Hz ShearwaterInc Glu Sugar moiety In vitro coupling —SH Cys Polymer, e.g.PEG-vinylsulphone Shearwater Inc Delgado et al, PEG, PEG-maleimidecritical reviews in Therapeutic Sugar moiety In vitro coupling DrugCarrier Systems 9(3, 4): 249-304 (1992) —OH Ser, Thr, OH—, Sugar moietyIn vivo O-linked Lys glycosylation —CONH₂ Asn as part of Sugar moiety Invivo an N-glycosylation glycosylation site Aromatic Phe, Tyr, Trp Sugarmoiety In vitro coupling residue —CONH₂ Gln Sugar moiety In vitrocoupling Yan and Wold, Biochemistry, 1984, Jul 31; 23(16): 3759-65Aldehyde Oxidized Polymer, e.g. PEG, PEGylation Andresz et al., 1978,Makromol. Ketone carbohydrate PEG-hydrazide Chem. 179: 301; WO 92/16555,WO 00/23114 Guanidino Arg Sugar moiety In vitro coupling Lundblad andNoyes, Chimical Reagents for Protein Modification, CRC Press Inc. BocaRaton, FI Imidazole ring His Sugar moiety In vitro coupling As forguanidine

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of theIFNG polypeptide is to be understood as one, two or all of the aminoacid residues constituting an N-glycosylation site is/are to be alteredin such a manner that either a functional N-glycosylation site isintroduced into the amino acid sequence, removed from said sequence or afunctional N-glycosylation site is retained in the amino acid sequence(e.g. by substituting a serine residue, which already constitutes partof an N-glycosylation site, with a threonine residue and vice versa).

In the present application, amino acid names and atom names (e.g. CA,CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) which are based on the IUPAC nomenclature(IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residuenames, atom names etc.), Eur. J. Biochem, 138, 9-37 (1984) together withtheir corrections in Eur. J. Biochem., 152, 1 (1985). CA is sometimesreferred to as Cα, CB as Cβ. The term “amino acid residue” is intendedto indicate an amino acid residue contained in the group consisting ofalanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D),glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine(Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Proor P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S),threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), andtyrosine (Tyr or Y) residues.

Numbering of amino acid residues in this document is from the N-terminusof wild-type human IFNG (huIFNG) without signal peptide (SEQ ID NO:1).

The terminology used for identifying amino acid positions/substitutionsis illustrated as follows: G18 indicates that position 18 is occupied bya glycine residue. G18N indicates that the Gly residue of position 18has been replaced with an Asn residue. Multiple substitutions areindicated with a “+”, e.g. G18N+S20T means an amino acid sequence whichcomprises a substitution of the Gly residue in position 18 with an Asnresidue and a substitution of the Ser residue in position 20 with a Thrresidue. Alternative substitutions are indicated with a “/”. Forexample, G18S/T covers the following individual substitutions: G18S andG18T. Deletions are indicated by an asterix. For example, G18* indicatesthat the Gly residue in position 18 has been deleted. Insertions areindicated the following way: Insertion of an additional Pro residueafter the Gln residue located at position 143 is indicated as Q143QP.Combined substitutions and insertions are indicated in the followingway: substitution of the Gln residue at position 143 with a Cys residueand insertion of a Pro residue after the position 143 amino acid residueis indicated as Q143CP.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence maybe of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “polymerase chain reaction” or “PCR” generally refers to amethod for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridising preferentially to atemplate nucleic acid.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.

“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide: a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

The term “modification”, as used herein, covers amino acidsubstitutions, amino acid insertions and amino acid deletions.

The terms “mutation” and “substitution” are used interchangeably herein.

The term “introduce” is primarily intended to mean substitution of anexisting amino acid residue, but may also mean insertion of anadditional amino acid residue.

The term “remove” is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

The term “one difference” or “differs from” as used in connection withspecific modifications is intended to allow for additional differencesbeing present apart from the specified amino acid modification. Thus, inaddition to the substitutions performed in the C-terminal part of theIFNG polypeptide aiming at controlling the C-terminal truncation of theIFNG polypeptide, the IFNG variant may, if desired, comprise othermodifications that are not related to this property. Such othermodifications may, for example, include introduction and/or removal ofamino acid residues comprising an attachment group for a non-polypeptidemoiety, addition of one or more extra residues at the N-terminus, e.g.addition of a Met residue at the N-terminus or addition of the aminoacid sequence Cys-Tyr-Cys at the N-terminus, as well as “conservativeamino acid substitutions”, i.e. substitutions performed within groups ofamino acids with similar characteristics, e.g. small amino acids, acidicamino acids, polar amino acids, basic amino acids, hydrophobic aminoacids and aromatic amino acids. Examples of conservative substitutionsin the present invention may, in particular, be selected from the groupslisted in the table below. 1 Alanine (A) Glycine (G) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Histidine (H) Lysine (K) 5 Isoleucine (I)Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y)Tryptophan (W)

The term “at least one” as used about a non-polypeptide moiety, an aminoacid residue, a substitution, etc. is intended to mean one or more.

The term “AUC_(sc)” or “Area Under the Curve when administeredsubcutaneously” is used in its normal meaning, i.e. as the area underthe IFNG activity in-serum-time curve, where the IFNG variant has beenadministered subcutaneously, in particular when administeredsubcutaneously in rats or non-human primates, such as monkeys. Once theexperimental IFNG activity-time points have been determined, theAUC_(sc) may conveniently be calculated by a computer program, such asGraphPad Prism 3.01.

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value. The functionalin vivo half-life may by determined in rats, cf. the Materials andMethod section herein, but is preferably determined in non-humanprimates, such as monkeys. It is important to note that the term“functional in vivo half-life”, when used herein, for a given IFNGvariant must be determined for a sample that has been administeredintravenously (iv).

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The serum half-life may bedetermined in rats, cf. the Materials and Method section herein, but ispreferably determined in non-human primates, such as monkeys. It isimportant to note that the term “serum half-life”, when used herein, fora given IFNG variant must be determined for a sample that has beenadministered intravenously (iv).

If not further specified, the terms “half-life” or “in vivo half-life”may refer to both functional in vivo half-life and serum half-life.

The term “serum” is used in its normal meaning, i.e. the term coversblood plasma without fibrinogen and other clotting factors.

The polypeptide is normally cleared by the action of one or more of thereticuloendothelial systems (RES), kidney, spleen or liver, or byspecific or unspecific proteolysis. The term “renal clearance” is usedin its normal meaning to indicate any clearance taking place by thekidneys, e.g. by glomerular filtration, tubular excretion or tubularelimination. Normally, renal clearance depends on physicalcharacteristics of the polypeptide, including molecular weight, size(relative to the cutoff for glomerular filtration), symmetry,shape/rigidity, charge and attached carbohydrate chains. A molecularweight of about 67 kDa is normally considered to be a cut-off-value forrenal clearance. Renal clearance may be measured by any suitable assay,e.g. an established in vivo assay. For instance, renal clearance may bedetermined by administering a labelled (e.g. radiolabelled orfluorescence labelled) conjugated polypeptide to a patient and measuringthe label activity in urine collected from the patient. Reduced renalclearance is determined relative to the reference molecule, such asglycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®. Thefunctionality to be retained is normally selected from antiviral,antiproliferative or immunomodulatory activity.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of theIFNG variant is statistically significantly increased relative to thatof a reference molecule, such as glycosylated huIFNG, glycosylated[S99T]huIFNG or Actimmune® determined under comparable conditions. Thus,interesting IFNG variants are such variants, which has an increasedfunctional in vivo half-life or an increased serum half-life as comparedto any of the reference molecules mentioned above, when administeredintravenously.

The term “reduced immunogenicity” is intended to indicate that the IFNGvariant gives rise to a measurably lower immune response than areference molecule, e.g. glycosylated huIFNG or Actimmune®, asdetermined under comparable conditions. The immune response may be acell or antibody mediated response (see, e.g., Roitt: EssentialImmunology (8th Edition, Blackwell) for further definition ofimmunogenicity). Normally, reduced antibody reactivity is an indicationof reduced immunogenicity. Reduced immunogenicity may be determined byuse of any suitable method known in the art, e.g. in vivo or in vitro.

In the present context the term “increased degree of in vivoN-glycosylation” or “increased degree of N-glycosylation” is intended toindicate increased levels of attached carbohydrate molecules, normallyobtained as a consequence of increased (or better) utilization ofglycosylation site(s). It is well-known (Hooker et al., 1998, J.Interferon and Cytokine Res. 18, 287-295 and Sarenva et al., 1995,Biochem J., 308, 9-14) that when huIFNG is expressed in CHO cells onlyabout 50% of the IFNG molecules utilizes both glycosylation sites, about40% utilizes one glycosylation site (1N), and about 10% is notglycosylated (0N). The increased degree of in vivo N-glycosylation maybe determined by any suitable method known in the art, e.g. by SDS-PAGE.One convenient assay for determining increased glycosylation is themethod described in the section entitled “Determination of IncreasedGlycosylation” in the Materials and Methods section herein.

The term “exhibiting IFNG activity” is intended to indicate that thevariant has one or more of the functions of native glycosylated huIFNGor Actimmune®, including the capability to bind to an IFNG receptor andcause transduction of the signal transduced upon huIFNG-binding of itsreceptor as determined in vitro or in vivo (i.e. in vitro or in vivobioactivity). The IFNG receptor has been described by Aguet et al. (Cell55:273-280, 1988) and Calderon et al. (Proc. Natl. Acad. Sci. USA85:4837-4841, 1988). A suitable assay for testing IFNG activity is theassay entitled “Primary Assay” disclosed herein. When using the “PrimaryAssay” described herein, polypeptide variants “exhibiting IFNG activity”have a specific activity of at least 5% as compared to glycosylatedhuIFNG, glycosylated [S99T]huIFNG or Actimamune®. It will be understoodthat depending on which specific modifications are performed, forexample whether the variant is PEGylated or not, this may lead toactivities over a wide range. Thus, examples of specific activities mayrange from as low as 5% to as high as 150% as compared to glycosylatedhuIFNG, glycosylated [S99T]huIFNG or Actimmune®. For example, thespecific activity may be at least 10% (e.g. 10-125%), such as at least15% (e.g. 15-125%), e.g. at least 20% (such as 20-125%), at least 25%(e.g. 25-125%), at least 30% (e.g. 30-125%), at least 35% (e.g.35-125%), at least 40% (e.g. 40-125%), at least 45% (e.g. 45-125%), atleast 50% (e.g. 50-125%), at least 55% (e.g. 55-125%), at least 60%(e.g. 60-125%), at least 65% (e.g. 65-125%), at least 70% (e.g.70-125%), at least 75% (e.g. 75-125%), at least 80% (e.g. 80-125%) or atleast 90% (e.g. 90-110%) as compared to the specific activity ofglycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®.

It may be beneficial that the variant has a decreased receptor-bindingaffinity and hence a decreased IFNG activity as compared to glycosylatedhuIFNG, glycosylated [S99T]huIFNG or Actimmune® in order to decreasereceptor-mediated clearence. For example, the variant may exhibit 1-75%(e.g. 5-75%), such as 1-50% (e.g. 5-50%), e.g. 1-40% (e.g. 5-40%), 1-30%(e.g. 5-30%), 1-20% (e.g. 5-20%) or 1-10% (e.g. 5-10%) of the IFNGactivity of glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimunne®when tested in the “Primary Assay” described herein.

An “IFNG polypeptide” is a polypeptide exhibiting IFNG activity, and isused herein about the polypeptide in monomer or dimeric form, asappropriate. For instance, when specific substitutions are indicatedthese are normally indicated relative to the IFNG polypeptide monomer.When reference is made to the IFNG polypeptide of the invention this isnormally in dimeric form (and thus, e.g., comprises two IFNG polypeptidemonomers modified as described). The dimeric form of the IFNGpolypeptides may be provided by the normal association of two monomersor be in the form of a single chain dimeric IFNG polypeptide. It will beunderstood that the IFNG polypeptides of the invention may, in additionto the specified modifications, also contain other modifications, e.g.modifications, which increases the AUC_(sc) and/or the functional invivo half-life of the IFNG polypeptide. Thus, the term “IFNGpolypeptide” also encompasses variant forms of the modified IFNGpolypeptides disclosed herein. Specific examples of such variants,which, in addition to substitutions in the C-terminal part of the IFNGpolypeptide, comprise further modifications include modifications suchas S99T and E38N+S40T. More examples of suitable modifications are givenbelow.

Normally, the variant forms of the IFNG polypeptide differs in 1-15 or2-15 amino acid residues (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acid residues), e.g. in 1-10 or 2-10 amino acidresidues, in 1-8 or 2-8 amino acid residues, in 1-5 or 2-5 amino acidresidues, or in 1-3 or 2-3 amino acid residues compared to huIFNG shownin SEQ ID NO:1.

The term “functional site” is intended to indicate one or more aminoacid residues which is/are essential for, or otherwise involved in, thefunction or performance of IFNG. Such amino acid residues are “locatedat” the functional site. The functional site may be determined bymethods known in the art and is preferably identified by analysis of astructure of the polypeptide complexed to a relevant receptor, such asthe IFNG receptor.

The term “huIFNG” is intended to mean the mature form of wild-type humanIFNG having the amino sequence shown in SEQ ID NO:1, including huIFNGproduced by recombinant means. If not further specified, the term“huIFNG” may refer to wild-type human IFNG in its glycosylated form(i.e. glycosylated at positions 25 and 97) or in its un-glycosylatedform.

When used herein the term “glycosylated” indicates that the IFNGpolypeptide is produced in a cell capable of glycosylating thepolypeptide and, therefore, the IFNG polypeptide is glycosylated at itsnative N-glycosylation sites (position 25 and 97 of SEQ ID NO:1).

When used herein the term “un-glycosylated” indicates that the IFNGpolypeptide is not glycosylated at its native N-glycosylation sites(position 25 and 97 of SEQ ID NO:1). Such “un-glycosylated” IFNGpolypeptides may be obtained by producing the polypeptide in aprokaryotic host cell, such as E. coli, not capable of glycosylation.

It will be understood that when the terms “glycosylated” and“un-glycosylated” are used about the variant IFNG polypeptides of theinvention, these terms indicate, independently of whether additionalglycosylation sites have been introduced and/or removed, whetherpotential glycosylation sites of the IFNG polypeptide variants areutilized or not.

In the present context the term “full-length” is intended to mean thatthe variant is not truncated as compared to the parent polypeptide,which is modified according to the invention. Preferably, the parentprotein to be modified as described herein is huIFNG. In this case theterm “full-length” means that the variant contains 143 amino acidresidues. Analogously, if the parent protein to be modified isMet-huIFNG, the term “full-length” mean that the variant contains 144amino acid residues.

When used herein, the term “C-terminal part” covers the last 12 aminoacid residues (calculated from the C-terminus) in huIFNG, i.e. aminoacid residues S132-Q143.

When used herein the term “Actimmune®” refers to the 140 amino acid formof IFNG (disclosed in SEQ ID NO:3) achieved by fermentation of agenetically engineered E. coli bacterium. Actimmune® is un-glycosylated.Further information of Actimmune® is available on www.actimmune.com.

In the present context the term “non-positively charged amino acidresidue” covers the following amino acid residues: A, V, L, I, M, F, W,P, G, S, T, C, Y, N, Q, D and E.

Interferon Gamma Variants of the Invention

Full-Length IFNG Variants

As indicated above, it has now been found that a substitution in eitherposition S132 or position S142 in combination with at least substitutionin a position selected from the group consisting of R137, R139 and R140,impedes the proteolytic processing of the polypeptide and hence resultsin a more homogenous product.

Thus, in a first aspect the present invention relates to a full-lengthIFNG polypeptide variant exhibiting IFNG activity, wherein said variantcomprises (a) at least one substitution in a position selected from thegroup consisting of S132 and S142; and (b) at least one amino acidsubstitution in a position selected from the group consisting of R137,R139 and R140.

Furthermore, the present invention also relates to a substantiallyhomogenous population of a full-length IFNG variant, wherein saidfull-length IFNG variant exhibits IFNG activity and wherein saidfull-length IFNG variant comprises (a) at least one substitution in aposition selected from the group consisting of S132 and S142; and (b) atleast one amino acid substitution in a position selected from the groupconsisting of R137, R139 and R140.

Moreover, the present invention also relates to a composition comprisinga substantially homogenous population of a full-length IFNG variant,wherein said full-length IFNG variant exhibits IFNG activity and whereinsaid full-length IFNG variant comprises (a) at least one substitution ina position selected from the group consisting of S132 and S142; and (b)at least one amino acid substitution in a position selected from thegroup consisting of R137, R139 and R140.

When used herein, the term “substantially homogenous population” isdefined as a population giving rise to a mass spectroscopic profilecharacterized by a single, dominant peak having an area under the curve(AUC) that is at least 3-fold higher than the AUC of any other peakappearing in the profile. Preferably, the AUC of the dominant peak is atleast 4-fold higher, such as at least 5-fold higher than the AUC of anyother peak appearing in the profile.

For example, the population of IFNG variants may contain at least 75% ofthe full-length IFNG variant of the invention (as compared to the totalamount of IFNG variants in the population), preferably at least 80%,such as at least 85%, e.g. at least 90%, more preferably at least 95%,such as at least 96%, e.g. at least 97%, even more preferably at least98%, such as at least 99%.

As will be understood the purpose of performing substitutions in one ormore of the positions R137, R139 or R140 is to change the furin proteasecleavage site and thereby avoiding proteolytic degradation of thepolypeptide. Many furin proteases recognise the sequenceR/K-X-R/K-R-↓-X, wherein X is any amino acid residue. Accordingly, R140may be substituted with any amino acid residue, including lysine, andR137 and R139 may be substituted with any amino acid residue, exceptlysine. Preferably, the amino acid residue to be introduced in one ormore of the positions R137, R139 and/or R140 is a non-positively chargedamino acid residue.

Amino acid residue to be introduced in one or more of the positionsR137, R139 and/or R140 may be selected from the group consisting ofsmall amino acid residues, such as Ala, Gly, Ser, Cys and Thr; acidicamino acid residues, such as Asp and Glu; hydrophobic amino acidresidues, such as Ile, Leu, Met, Pro and Val; aromatic amino acidresidues, such as Phe, Trp and Tyr; and polar amino acid residues, suchas Asn and Gln. In a highly preferred embodiment of the invention theamino acid residue to be introduced in one or more of the positionsR137, R139 and/or R140 is a proline residue.

Amino acid residue to be introduced in position S132 or S142 may beselected from the group consisting of small amino acid residues, such asAla, Gly, Ser, Cys and Thr; acidic amino acid residues, such as Asp andGlu: hydrophobic amino acid residues, such as Ile, Leu, Met, Pro andVal; aromatic amino acid residues, such as Phe, Trp and Tyr; and polaramino acid residues, such as Asn and Gln. In a highly preferredembodiment of the invention the amino acid residue to be introduced inposition S132 or S142 is Pro. Accordingly, in a preferred embodiment ofthe invention, the variant comprises a substitution selected from thegroup consisting of S132P, S142P and S132P+S142P, in particular S132P orS142P.

As explained above, the C-terminal part of the IFNG variant should, inaddition to a substitution in position S132 and/or S142, contain atleast one further substitution in a position selected from the groupconsisiting of R137, R139, R140 and combinations thereof. Thus, inaddition to a substitution in position S132 and/or S142, the C-terminalpart of the variant shall contain a substitution in a position selectedfrom the group consisting of R137, R139, R140, R137+R139, R137+R140,R139+R140 and R137+R139+R140.

Specific examples of substitutions in posititon R137 include R137A,R137V, R137L, R137I, R137M, R137F, R137W, R137P, R137G, R137S, R137T,R137C, R137Y, R137N, R137Q, R137D and R137E, preferably R137P.

Specific examples of substitutions in posititon R139 include R139A,R139V, R139L, R139I, R139M, R139F, R139W, R139P, R139G, R139S, R139T,R139C, R139Y, R139N, R139Q, R139D and R139E, preferably R139P.

Specific examples of substitutions in posititon R140 include R140A,R140V, R140L, R140I, R140M, R140F, R140W, R140P, R140G, R140S, R140T,R140C, R140Y, R140N, R140Q, R140D, R140E and R140K, preferably R140P.

Preferably, the C-terminal part of the variant contains a substitutionin a position selected from the group consisting of R137P, R139P, R140P,R137P+R139P, R137P+R140P, R139P+R140P and R137P+R139P+R140P.

Specific examples of full-length IFNG variants comprising a substitutionin position S132 include variants selected from the group consisting ofS132P+R137P, S132P+R139P, S132P+R140P, S132P+R137P+R139P,S132P+R137P+R140P, S132P+R139P+R140P and S132P+R137P+R139P+R140P,preferably selected from the group consisting of S132P+R137P+R140P andS132P+R140P, most preferably S132P+R137P+R140P.

Specific examples of full-length IFNG variants comprising a substitutionin position S142 include variants selected from the group consisting ofR137P+S142P, R139P+S142P, R140P+S142P, R137P+R139P+S142P,R137P+R140P+S142P, R139P+R140P+R142P and R137P+R139P+R140P+S142P,preferably selected from the group consisting of R137P+S142P andR137P+R139P+S142P.

As also indicated above, it has also now been found that a substitutionin positions R137 and R140 impairs the proteolytic processing of thepolypeptide and hence results in a more homogenous product.

Thus, in a second aspect the present invention relates to a full-lengthIFNG polypeptide variant exhibiting IFNG activity, wherein said variantcomprises an amino acid substitution in position R137 and an amino acidsubstitution in position R140.

Furthermore, the present invention also relates to a substantiallyhomogenous population of a full-length IFNG variant, wherein saidfull-length IFNG variant exhibits IFNG activity and wherein saidfull-length IFNG variant comprises an amino acid substitution inposition 137 and an amino acid substitution in position R140.

Moreover, the present invention also relates to a composition comprisinga substantially homogenous population of a full-length IFNG variant,wherein said full-length IFNG variant exhibits IFNG activity and whereinsaid full-length IFNG variant comprises an amino acid substitution inposition R137 and an amino acid substitution in position R140.

When used herein, the term “substantially homogenous population” isdefined as a population giving rise to a mass spectroscopic profilecharacterized by a single, dominant peak having an area under the curve(AUC) that is at least 3-fold higher than the AUC of any other peakappearing in the profile. Preferably, the AUC of the dominant peak is atleast 4-fold higher, such as at least 5-fold higher than the AUC of anyother peak appearing in the profile.

For example, the population of IFNG variants may contain at least 75% ofthe full-length IFNG variant of the invention (as compared to the totalamount of IFNG variants in the population), preferably at least 80%,such as at least 85%, e.g. at least 90%, more preferably at least 95%,such as at least 96%, e.g. at least 97%, even more preferably at least98%, such as at least 99%.

Amino acid residue to be introduced in the positions R137 and R140 mayindependently be selected from the group consisting of small amino acidresidues, such as Ala, Gly, Ser, Cys and Thr; acidic amino acidresidues, such as Asp and Glu; hydrophobic amino acid residues, such asIle, Leu, Met, Pro and Val; aromatic amino acid residues, such as Phe,Trp and Tyr; and polar amino acid residues, such as Asn and Gln.

In a preferred embodiment of the invention at least one of the aminoacid residues to be introduced in the positions R137 and R140 is Pro.

Thus, in a preferred embodiment of the invention, the variant comprisesthe substitutions R137X+R140P, wherein X is any amino acid residue,except arginine and lysine.

In another preferred embodiment of the invention, the variant comprisesthe substitutions R137P+R140X, wherein X is any amino acid residueexcept arginine.

In a highly preferred embodiment of the invention the amino acid residueto be introduced in both of positions R137 and R140 is Pro, i.e. in ahighly preferred embodiment of the invention, the variant comprises thesubstitutions R137P+R140P.

Further Modifications in the C-Terminal Part

The above-mentioned modifications may be the only modifications in theC-terminal part of the variant. However, in another embodiment of theinvention the variant comprises at least one further modification in theC-terminal part of the variant from amino acid residue S132 to aminoacid Q143. For example, as will be acknowledged by the skilled person,introduction of a non-naturally occurring residue in a human polypeptidemay give rise to an epitope capable of inducing a response from thehuman immune system.

This problem may be effectively solved by “shielding” the introducedresidues, e.g. by introducing an in vivo N-glycosylation site or othernon-polypeptide moieties, such as PEG, in the vicinity of the introducedresidues. For example, an amino acid residue comprising an attachmentgroup for a non-polypeptide moiety (which is capable of screening thepotential epitope) may be introduced in the vicinity of the introducedresidues. One particular preferred amino acid residue comprising anattachment group for a non-polypeptide moiety, such as PEG, is cysteine.Apart from shielding the introduced residues, the introduced cyeteineresidue may, when covalently attached to a non-polypeptide moiety, suchas PEG, confer additional advantageous properties to the IFNGpolypeptide, such as increased functional in vivo half-life and/orincreased AUC_(sc).

Although introduction of N-glycosylation sites in the C-terminal part ofthe IFNG polypeptide is also contemplated according to the presentinvention, this approach is not particularly preferred since fullutilization of N-glycosylation sites in the C-terminal part of IFNG mayprove difficult.

Thus, in another embodiment of the invention the variant furthercomprises at least one cysteine residue in the C-terminal part of thevariant from amino acid residue S132 to amino acid Q143. Specificexamples of substitutions which introduce a cysteine residue in theC-terminal part of the IFNG variant include substitutions selected fromthe group consisting of S132C, Q133C, M134C, L135C, F136C, R137C, G138C,R139C, R140C, A141C, S142 and Q143. It will be understood that cysteineresidues should not be introduced in positions which are essential forobtaining the full-length IFNG gamma variant.

Although two or more cysteine residues may be introduced in theC-terminal part of the IFNG variant it is preferred that only a singlecysteine residue is introduced in this region of the molecule.

As will be understood, the introduced cysteine residue is preferablycovalently attached (conjugated) to a non-polypeptide moiety, such as apolymer molecule, preferably PEG or more preferably mPEG having amolecular weight from 1-20 kDa, such as 1 kDa, 2 kDa, 5 kDa, 10 kDa, 12kDa or 20 kDa. The conjugation between the cysteine-containing IFNGpolypeptide and the polymer molecule may be achieved in any suitablemanner, e.g. as described in the section entitled “Conjugation to apolymer molecule”, e.g. in using a one step method or in the stepwisemanner referred to in said section. The preferred method for PEGylatingthe IFNG polypeptide is to covalently attach PEG to cysteine residuesusing cysteine-reactive PEGs. A number of highly specific,cysteine-reactive PEGs with different groups (e.g. maleimide,vinylsulfone and orthopyridyl-disulfide) and different size PEGs (2-20kDa) are commercially available, e.g. from Shearwater Polymers Inc.,Huntsville, Ala., USA.

Modifications in the Amino Acid Sequence from Residue No. 1 to ResidueNo. 131

As far as the amino acid sequence from residue no. 1 to residue no. 131is concerned, this part of the sequence may be identical to residue no.1 to residue no. 131 of huIFNG and may be glycosylated (e.g. byproducing the variant in a glycosylation host cell) or un-glycosylated(e.g. by producing the variant in a prokaryotic host cell, such as E.coli).

However, in a preferred embodiment of the invention, the variantcomprises an amino acid sequence from residue no. 1 to residue no. 131,which further comprises 1-10, such as 1-7, e.g. 1-5 or 1-3modifications, preferably substitutions, compared to amino acid residueno. 1 to residue no. 131 of huIFNG. One example includes thesubstitution S99T (leading to a more efficient utilization of theposition 97 N-glycosylation site). Other interesting substitutionsinclude E38N+S40T (leading to an increased AUC_(sc)), in particularE38N+S40+S99T.

Such variants may be glycosylated or un-glycosylated. Preferably, suchvariants are glycosylated.

Other interesting modifications are discussed in the below sectionsentitled “IFNG variants with optimised N-glycosylation sites”, “IFNGvariants with increased AUC_(sc) and/or increased half-life”, “IFNGvariants wherein the non-polypeptide moiety is a sugar moiety”, “IFNGvariants wherein the non-polypeptide moiety is a molecule, which hascysteine as an attachment group” and “IFNG variants wherein the firstnon-polypeptide moiety is a sugar moiety and the second non-polypeptidemoiety is a molecule, which has cysteine as an attachment group”.

IFNG Variants with Optimised N-Glycosylation Sites

As mentioned above, the amino acid sequence from residue no. 1 toresidue no. 131 preferably comprises 1-10, such as 1-7, e.g. 1-5 or 1-3,modifications, preferably substitutions, compared to amino acid residueno. 1 to residue no. 131 of huIFNG.

One class of interesting modifications that may be introduced into thispart of the sequence include modifications, which serve to optimise theglycosylation of a given glycosylation site.

It has been found (see WO 02/081507) that glycosylation of the naturallyoccurring N-glycosylation site located in position 97 of huIFNG may beincreased, i.e. an increased fraction of fully, or substantially fully,glycosylated IFNG polypeptides may be obtained, by substituting theserine residue located in position 99 of huIFNG with a threonineresidue. For example, by performing the S99T substitution it has beenfound that about 90% of the polypeptides present in the harvested mediumutilize both N-glycosylation sites, whereas only about 60% of the huIFNGpolypeptides present in the harvested medium were fully glycosylated.

Accordingly, in a very interesting embodiment of the invention, the IFNGvariant of the invention comprises the substitution S99T.

In addition to the already mentioned S99T mutation required foroptimisation of the in vivo N-glycosylation site at position 97, otherin vivo glycosylation sites, which may have been introduced into thesequence (see the section entitled “IFNG variants wherein thenon-polypeptide moiety is a sugar moiety”) may be optimised. Normally,the in vivo glycosylation site is an N-glycosylation site, but also anO-glycosylation site is contemplated as relevant for the presentinvention. This optimisation may be achieved by performing amodification, preferably a substitution, in a position, which is locatedclose to a glycosylation site, in particular close to an in vivoN-glycosylation site. Specific examples of suitable positions where invivo N-glycosylation sites may be introduced, are disclosed in WO01/36001, WO 02/081507 and in the section entitled “IFNG variantswherein the non-polypeptide moiety is a sugar moiety”.

An amino acid residue “located close to” a glycosylation site is usuallylocated in position −4, −3, −2, −1, +1, +2, +3 or +4 relative to theamino acid residue of the glycosylation site to which the carbohydrateis attached, preferably in position −1, +1, or +3, in particular inposition +1 or +3. Thus, the amino acid residue located close to an invivo N-glycosylation site (having the sequence N-X-S/T/C) may be locatedin position −4, −3, −2, −1, +1, +2, +3 or 4 relative to the N-residue.

When position +2 relative to the N-residue is modified it will beunderstood that only a limited number of modifications are possiblesince in order to maintain/introduce an in vivo N-glycosylation site,the amino acid residue in said position must be either Ser, Thr or Cys.In a particular preferred embodiment of the invention, the modificationof the amino acid residue in position +2 relative to the in vivoN-glycosylation site is a substitution where the amino acid residue inquestion is replaced with a Thr residue. If, on the other hand, saidamino acid residue is already a Thr residue it is normally not preferredor necessary to perform any substitutions in that position. When X ismodified, X should not be Pro and preferably not Trp, Asp, Glu and Leu.If X is modified, the amino acid residue to be introduced is preferablyselected form the group consisting of Phe, Asn, Gln, Tyr, Val, Ala, Met,Ile, Lys, Gly, Arg, Thr, His, Cys and Ser, more preferably Ala, Met,Ile, Lys, Gly, Arg, Thr, His, Cys and Ser, in particular Ala or Ser.When position +3 relative to the N-residue is modified, the amino acidresidue to be introduced is preferably selected from the groupconsisting of His, Asp, Ala, Met, Asn, Thr, Arg, Ser and Cys, morepreferably Thr, Arg, Ser and Cys. Such modifications are particularrelevant if the X residue is a Ser residue.

Thus, with respect to the naturally present in vivo N-glycosylation, itis contemplated that the N-glycosylation site at position 97 may befurther optimised by performing a modification, such as a substitution,in a position selected from the group consisting of E93, K94, L95, T96,Y98, V100 and T101 (i.e. at position 4, −3, −2, −1, +1, +3 or +4relative to N97). Specific examples of substitutions performed inposition 98 include Y98F, Y98N, Y98Q, Y98V, Y98A, Y98M, Y98I, Y98K,Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, preferably Y98A, Y98M, Y98I,Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, in particular Y98S.Specific examples of substitutions performed in position 100 includeV100H, V100D, V100A, V100M, V100N, V100T, V100R, V100S, or V100C, inparticular V100T, V100R, V100S or V100C.

In a similar way, with respect to the in vivo N-glycosylation site atposition 25 it is contemplated that this site may be further optimisedby performing a modification, such as a substitution, in a positionselected from the group consisting of D21, V22, A23, D24, G26, L28 andF29 (i.e. at position −4, −3, −2, −1, +1, +3 or +4 relative to N25).Specific exam substitutions performed in position 26 include G26F, G26N,G26Y, G26Q, G26V, G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C andG26S, preferably G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C andG26S, more preferably G26A and G26S, in particular G26A. Specificexamples of substitutions performed in position 28 include G28H, G28D,G28A, G28M, G28N, G28T, G28R, G28S, or G28S, in particular G28A, G28T,G28R, G28R, G28S or G28C.

Obviously, any of the modifications mentioned in connection withoptimisation of glycosylation at position 97 may be combined with any ofthe mentioned in connection with optimisation of glycosylation atposition 25.

IFNG Variants with Increased AUC_(sc) and/or Increased Half-Life

Another class of interesting modifications that may be introduced intothe amino acid sequence from residue no. 1 to residue no. 131 includemodifications, in particular substitutions, which serve to increase theAUC_(sc) and/or the serum half-life/functional in vivo half-life whenadministered intravenously.

In a particular interesting embodiment of the invention the IFNG variantcomprises, in the amino acid sequence from residue no. 1 to residue no.131, at least one introduced and/or at least one removed amino acidresidue comprising an attachment group for a non-polypeptide moiety.Preferably, the amino acid sequence from residue no. 1 to residue no.131 comprises at least one introduced amino acid residue comprising anattachment group for a non-polypeptide moiety.

Such variants typically exhibit an increased functional in vivohalf-life and/or an increased AUC_(sc).

Thus, interesting IFNG variants are such variants where the ratiobetween the serum half-life (or functional in vivo half-life) of saidvariant and the serum half-life (or functional in vivo half-life) ofglycosylated huIFNG or glycosylated [S99T]huIFNG is at least 1.25, morepreferably at least 1.50, such as at least 1.75, e.g. at least 2, evenmore preferably at least 3, such as at least 4, e.g. at least 5, whenadministered intravenously, in particular when administeredintraveniously in non-human primates, such as monkeys.

Other examples of interesting IFNG variants are such variants where theratio between the serum half-life (or functional in vivo half-life) ofsaid variant and the serum half-life (or functional in vivo half-life)of Actimmune® is at least 2 more preferably at least 3, such as at least4, e.g. at least 5, even more preferably at least 6, such as at least 7,e.g. at least 8, most preferably at least 9, such as at least 10, whenadministered intravenously, in particular when administeredintraveniously into rats or non-human primates, such as monkeys.

The term “increased” as used about the AUC_(sc) is used to indicate thatthe Area Under the Curve for an IFNG variant of the invention, whenadministered subcutaneously, is statistically significantly increasedrelative to that of a reference molecule, such as glycosylated huIFNG,glycosylated [S99T]huIFNG or Actimmune®, determined under comparableconditions. Thus, preferred IFNG variants are such variants, which havean increased AUC_(sc), as compared to any of the reference moleculesmentioned above. Evidently, the same amount of IFNG activity should beadministered for the IFNG variant of the invention and the referencemolecule.

Particular preferred IFNG variants are such variants where the ratiobetween the AUC_(sc) of said variant and the AUC_(sc) of glycosylatedhuIFNG or glycosylated [S99T]huIFNG is at least 1.25, such as at least1.5, e.g. at least 2, more preferably at least 3, such as at least 4,e.g. at least 5 or at least 6, even more preferably at least 7, such asat least 8, e.g. at least 9 or at least 10, most preferably at least 12,such as at least 14, e.g. at least 16, at least 18 or at least 20, inparticular when administered (subcutaneously) in rats.

Other examples of particular preferred IFNG variants are such variantswhere the ratio between the AUC_(sc) of said variant and the AUC_(sc) ofActimmune® is at least 100, more preferably at least 150, such as atleast 200, e.g. at least 250, even more preferably at least 300, such asat least 400 e.g. at least 500, most preferably at least 750, such as atleast 1000, e.g. at least 1500 or at least 2000, in particular whenadministered (subcutaneously) in rats.

In order to avoid too much disruption of the structure and function ofthe IFNG polypeptide the total number of amino acid residues to bemodified in accordance with this embodiment of the invention (i.e. inthe region from residue no. 1 to residue no. 131) typically does notexceed 10. Usually the amino acid sequence from residue no. 1 to residueno. 131 comprises 1-10, such as 1-7, e.g. 1-5 or 1-3 modificationscompared to residue no. 1 to residue no. 131 of huIFNG. Thus, normallythe IFNG variant comprises an amino acid sequence (from residue no. 1 toresidue no. 131) which differs from the amino acid sequence of huIFNG(from residue no. 1 to residue no. 131) in 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid residues. Preferably, the modification(s) is/are asubstitution(s).

By removing or introducing an amino acid residue comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimse the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the IFNG polypeptide) andthereby obtain a new conjugate molecule, which exhibits IFNG activityand in addition one or more improved properties as compared to thehuIFNG based molecules available today. For instance, by introduction ofattachment groups, the IFNG polypeptide is boosted or otherwise alteredin the content of the specific amino acid residues to which the relevantnon-polypeptide moiety binds, whereby a more efficient, specific and/orextensive conjugation is achieved. By removal of one or more attachmentgroups it is possible to avoid conjugation to the non-polypeptide moietyin parts of the polypeptide in which such conjugation isdisadvantageous, e.g. to an amino acid residue located at or near afunctional site of the polypeptide (since conjugation at such a site mayresult in inactivation of the resulting conjugated polypeptide due toimpaired receptor recognition). Further, it may be advantageous toremove an attachment group located closely to another attachment groupin order to avoid heterogeneous conjugation to such groups. In aninteresting embodiment more than one amino acid residue of the IFNGpolypeptide is altered, e.g. the alteration embraces removal as well asintroduction of amino acid residues comprising attachment sites for thenon-polypeptide moiety of choice. This embodiment is considered ofparticular interest in that it is possible to specifically design theIFNG polypeptide so as to obtain an optimal conjugation to thenon-polypeptide moiety.

In addition to the removal and/or introduction of amino acid residues,the polypeptide may comprise other modifications, e.g. substitutions,that are not related to introduction and/or removal of amino acidresidues comprising an attachment group for the non-polypeptide moiety.Examples of such modifications include conservative amino acidsubstitutions and/or introduction of Cys-Tyr-Cys or Met at theN-terminus.

The exact number of attachment groups available for conjugation andpresent in the IFNG polypeptide in dimeric form is dependent on theeffect desired to be achieved by the conjugation. The effect to beobtained is, e.g., dependent on the nature and degree of conjugation(e.g. the identity of the non-polypeptide moiety, the number ofnon-polypeptide moieties desirable or possible to conjugate to thepolypeptide, where they should be conjugated or where conjugation shouldbe avoided, etc.).

It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, either it be removed orintroduced, is selected on the basis of the nature of thenon-polypeptide moiety part of choice and, in most instances, on thebasis of the conjugation method to be used. For instance, when thenon-polypeptide moiety is a polymer molecule such as a polyethyleneglycol- or polyalkylene oxide-derived molecule, amino acid residuescapable of functioning as an attachment group may be selected from thegroup consisting of cysteine, lysine, aspartic acid, glutamic acid andarginine. In particular, cysteine is preferred. When the non-polypeptidemoiety is a sugar moiety the attachment group is, e.g., an in vivoglycosylation site, preferably an N-glycosylation site.

Whenever an attachment group for a non-polypeptide moiety is to beintroduced into or removed from the IFNG polypeptide, the position ofthe polypeptide to be modified is conveniently selected as follows:

The position is preferably located at the surface of the IFNGpolypeptide, and more preferably occupied by an amino acid residue thathas more than 25% of its side chain exposed to the surface, inparticular more than 50% of its side chain exposed to the surface, asdetermined on the basis of a 3D structure or model of IFNG in itsdimeric form, the structure or model optionally further comprising oneor two IFNG receptor molecules. Such positions are listed in Example 1herein.

In addition, it may be of interest to modify one or more amino acidresidues located in the loop regions of IFNG since most amino acidresidues within these loop regions are exposed to the surface andlocated sufficiently far away from functional sites so thatnon-polypeptide moieties, such as polymer molecules, in particular PEGmolecules, and/or N-glycosylation sites, may be introduced withoutimpairing the function of the molecule. Such loops regions may beidentified by inspection of the three-dimensional structure of huIFNG,optinally in complex with its receptor(s). The amino acid residuesconstituting said loop regions are residues N16-K37 (the “A-B loop”),F60-S65 (the “B-C loop”), N83-S84 (the “C-D loop”) and Y98-L103 (the“D-E loop”).

Furthermore, in the IFNG variants of the invention, attachment groupslocated at the receptor-binding site of IFNG may be removed, preferablyby substitution of the amino acid residue comprising such group. Theamino acid residues constituting the IFNG receptor-binding site are Q1,D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26, T27,L30, K34, K37, K108, H111, E112, I114, Q115, A118 and E119 (see alsoExample 2 herein).

In the case of a single chain IFNG polypeptide it may be sufficient toremove attachment groups in the receptor-binding site of only one of themonomers and thereby obtain a single chain IFNG polypeptide conjugatewith one active and one inactive receptor-binding site.

In order to determine an optimal distribution of attachment groups, thedistance between amino acid residues located at the surface of the IFNGpolypeptide is calculated on the basis of a 3D structure of the IFNGdimeric polypeptide. More specifically, the distance between the CB's ofthe amino acid residues comprising such attachment groups, or thedistance between the functional group (NZ for lysine, CG for asparticacid, CD for glutamic acid, SG for cysteine) of one and the CB ofanother amino acid residue comprising an attachment group aredetermined. In case of glycine, CA is used instead of CB. In the IFNGpolypeptide part of the invention any of said distances is preferablymore than 8 Å, in particular more than 10 Å in order to avoid or reduceheterogeneous conjugation.

Also, the amino acid sequence of the IFNG polypeptide variant may differfrom the huIFNG amino acid sequence in that one or more amino acidresidues constituting part of an epitope has been removed, preferably bysubstitution to an amino acid residue comprising an attachment group forthe non-polypeptide moiety, so as to destroy or inactivate the epitope.Epitopes of huIFNG may be identified by use of methods known in the art,also known as epitope mapping, see, e.g. Romagnoli et al., Biol Chem,1999, 380(5):553-9, DeLisser H M, Methods Mol Biol, 1999, 96:11-20, Vande Water et al., Clin Immunol Immunopathol, 1997, 85(3):229-35,Saint-Remy J M, Toxicology, 1997, 119(1):77-81, and Lane D P and StephenC W, Curr Opin Immunol, 1993, 5(2):268-71. One method is to establish aphage display library expressing random oligopeptides of e.g. 9 aminoacid residues. IgG1 antibodies from specific antisera towards huIFNG arepurified by immunoprecipitation and the reactive phages are identifiedby immunoblotting. By sequencing the DNA of the purified reactivephages, the sequence of the oligopeptide can be determined followed bylocalization of the sequence on the 3D-structure of the IFNG. Thethereby identified region on the structure constitutes an epitope thatthen can be selected as a target region for introduction of anattachment group for the non-polypeptide moiety.

Functional in vivo half-life and serum half-life is inter alia dependenton the molecular weight of the polypeptide and the number of attachmentgroups needed for providing increased half-life thus depends on themolecular weight of the non-polypeptide moiety in question. In oneembodiment, the IFNG polypeptide of the invention has a molecular weightof at least 67 kDa, in particular at least 70 kDa as measured bySDS-PAGE according to Laemmli, U. K., Nature Vol. 227 (1970), p. 680-85.IFNG has a MW in the range of about 34-50 kDa, and therefore additionalabout 20-40 kDa is required to obtain the desired effect. This may,e.g., be provided by 2-4 10 kDa PEG molecules or by a combination ofadditional in vivo glycosylation sites and additional PEG molecules, oras otherwise described herein.

Preferably, a conjugated IFNG polypeptide according to the inventioncomprises 1-10 (additional) non-polypeptide moieties, such as 1-8, 2-8,1-5 or 2-5 (additional) non-polypeptide moieties. Typically, thepolypeptide will comprise 1-3 (additional) non-polypeptide moieties,such as 1, 2 or 3 (additional) non-polypeptide moieties.

As mentioned above, under physiological conditions IFNG exists as adimeric polypeptide. The polypeptide is normally in homodimeric form(e.g. prepared by association of two IFNG polypeptide molecules preparedas described herein). However, if desired the IFNG polypeptide may beprovided in single chain form, wherein two IFNG polypeptide monomers arelinked via a peptide bond or a peptide linker. Providing the IFNGpolypeptide in single chain form has the advantage that the twoconstituent IFNG polypeptides may be different which can beadvantageous, e.g., to enable asymmetric mutagenesis of thepolypeptides. For instance, PEGylation sites can be removed from thereceptor-binding site from one of the monomers, but retained in theother. Thereby, after PEGylation one monomer has an intactreceptor-binding site, whereas the other may be fully PEGylated (andthus provide significantly increased molecular weight).

IFNG Variants Wherein the Non-Polypeptide Moiety is a Sugar Moiety

In a particular preferred embodiment of the invention, the IFNG variantcomprises, in the amino acid sequence from residue no. 1 to residue no.131, at least one introduced and/or at least one removed glycosylationsite, i.e. the non-polypeptide moiety is a sugar moiety. Preferably, theglycosylation site is an in vivo glycosylation site, e.g. an O-linked orN-linked sugar moiety, preferably an N-linked sugar moiety.

Thus, in one interesting embodiment of the invention said IFNG variantcomprises, in the amino acid sequence from residue no. 1 to residue no.131, at least one introduced glycosylation site, in particular anintroduced in vivo N-glycosylation site. Preferably, the introducedglycosylation site is introduced by a substitution.

For instance, an in vivo N-glycosylation site may be introduced into aposition (from residue no. 1 to residue no. 131) of the IFNG variantcomprising an amino acid residue exposed to the surface. Preferably saidsurface-exposed amino acid residue has at least 25% of the side chainexposed to the surface, in particular at least 50% of its side chainexposed to the surface. Details regarding determination of suchpositions can be found in Example 1 herein.

The N-glycosylation site is introduced in such a way that the N-residueof said site is located in said position. Analogously, anO-glycosylation site is introduced so that the S or T residue making upsuch site is located in said position.

It should be understood that when the term “at least 25% (or 50%) of itsside chain exposed to the surface” is used in connection withintroduction of an in vivo N-glycosylation site this term refers to thesurface accessibility of the amino acid side chain in the position wherethe sugar moiety is actually attached. In many cases it will benecessary to introduce a serine or a threonine residue in position +2relative to the asparagine residue to which the sugar moiety is actuallyattached and these positions, where the serine or threonine residues areintroduced, are allowed to be buried, i.e. to have less than 25% (or50%) of their side chains exposed to the surface of the molecule.

Furthermore, in order to ensure efficient glycosylation it is preferredthat the in vivo glycosylation site, in particular the N residue of theN-glycosylation site or the S or T residue of the O-glycosylation site,is located within the 118 N-terminal amino acid residues of the IFNGpolypeptide, more preferably within the 97 N-terminal amino acidresidues. Still more preferably, the in vivo glycosylation site isintroduced into a position wherein only one mutation is required tocreate the site (i.e. where any other amino acid residues required forcreating a functional glycosylation site is already present in themolecule).

For instance, substitutions that lead to introduction of an additionalN-glycosylation site at positions exposed at the surface of the IFNGpolypeptide and occupied by amino acid residues having at least 25% ofthe side chain exposed to the surface (in a structure with receptormolecule) include: Q1N+P3S/T, P3N+V5S/T, K6N+A8S/T, E9N+L11S/T, K12S/T,K13N+F15S/T, Y14N+N16S/T, G18S/T, G18N, G18N+S20T, H19N+D21S/T,D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37S/T, K37N+E39S/T,E38N, E38N+S40T, E39N+D41S/T, S40N+R42S/T, K55N+F57S/T, K58N+F60S/T,K61S/T, K61N+D63S/T, D62N+Q64S/T, D63N, D63N+S65T, Q64N+I66S/T,S65N+Q67S/T, Q67N, Q67N+S69T, K68N+V70S/T, E71N+I73S/T, T72N+K74S/T,K74N+D76S/T, E75N+M77S/T, K80S/T, V79N+F81S/T, K80N+F82S/T, N85S/T,S84N+K86S/T, K87S/T, K86N+K88S/T, K87N+R89S/T, D90N+F92S/T, E93N+L95S/T,K94N, K94N+T96S, T101N+L103S/T, D102N+N104S/T, L103N+V105S/T, Q106S/T,E119N, E119N+S121T, P122N+A124S/T, A123N+K125S/T, A124N, A124N+T126S,K125N+G127S/T, T126N+K128S/T, G127N+R129S/T, K128N+K130S/T,R129N+R131S/T and K130N. S/T indicates a substitution to a serine orthreonine residue, preferably a threonine residue.

Substitutions that lead to introduction of an additional N-glycosylationsite at positions exposed at the surface of the IFNG polypeptide havingat least 50% of the side chain exposed to the surface (in a structurewith receptor molecule) include: P3N+V5S/T, K6N+A8S/T, K12S/T,K13N+F15S/T, G18S/T, D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T,K37N+E39S/T, E38N, E38N+S40S/T, E39N+D41S/T, K55N+F57S/T, K58N+F60S/T,K61S/T, D62N+Q64S/T, Q64N+I66S/T, S65N+Q67S/T, K68N+V70S/T, E71N+I73S/T,E75N+M77S/T, N85S/T, S84N+K86S/T, K86N+K88S/T, K87N+R89S/T, K94N,K94N+T96S, T101N+L103S/T, D102N+N104S/T, L103N+V105S/T, Q106S/T,P122N+A124S/T, A123N+K125S/T, A124N, A124N+T126S, K125N+G127S/T,T126N+K128S/T, G127N+R129S/T, K128N+K130S/T, R129N+R131S/T, K130N andK130N+S132T. S/T indicates a substitution to a serine or threonineresidue, preferably a threonine residue.

Substitutions where only one amino acid substitution is required tointroduce an N-glycosylation site include K12S/T, G18S/T, G18N, K37S/T,E38N, M45N, I49N, K61S/T, D63N, Q67N, V70N, K80S/T, F82N, N85S/T,K87S/T, K94N, Q106S/T, E119N, A124N, K130N and R140N, in particularG18N, G18S/T, K37S/T, E38N, K61S/T, D63N, Q67N, K80S/T, N85S/T, K94N,Q106S/T, A124N and K130N (positions with more than 25% of its site chainexposed to the surface in a structure without receptor molecule), ormore preferably G18N, E38N, D63N, Q67N, K94N, A124N and K130N (positionswith more than 50% of its side chain exposed to the surface in astructure without receptor molecule).

Usually, it is not preferred to introduce N-glycosylation sites in theregion constituting the receptor binding site (except in special cases,cf. the section entitled “Variants with a reduced receptor affinity”).Accordingly, the mutations Q1N+P3S/T, E9N+L11S/T, G18N, G18N+S20T,H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, K34N+W36S/T, K37N+E39S/T, E119Nand E119N+S121T should normally not be performed, unless a reducedreceptor affinity is desired. In addition, the positive cluster K128,R129, K130 and R131 is required for activity and should normally not bemodified.

Particular preferred IFNG variants of invention include at least onefurther substitution selected from the group consisting of G18S, G18T,E38N, E38N+S40T, K61S, K61T, S65N+Q67S, S65N+Q67T, N85S, N85T, K94N,Q106S and Q106T, more preferably selected from the group consisting ofG18T, E38N+S40T, K61T, S65N+Q67T, N85T, K94N and Q106T, even morepreferably selected from the group consisting of G18T, E38N+S40T, K61T,S65N+Q67T and N85T, in particular E38N+S40T.

Thus, specific examples of interesting full-length variants of theinvention include variants selected from the group consisting of

-   [E38N+S40T+S99T+S132P+R137P]huIFNG,-   [E38N+S40T+S99T+S132P+R139P]huIFNG,-   [E38N+S40T+S99T+S132P+R140P]huIFNG,-   [E38N+S40T+S99T+S132P+R137P+R139P]huIFNG,-   [E38N+S40T+S99T+S132P+R137P+R140P]huIFNG,-   [E38N+S40T+S99T+S132P+R139P+R140P]huIFNG,-   [E38N+S40T+S99T+S132P+R137P+R139P+R140P]huIFNG,-   [E38N+S40T+S99T+R137P+S142P]huIFNG,-   [E38N+S40T+S99T+R139P+S142P]huIFNG,-   [E38N+S40T+S99T+R140P+S142P]huIFNG,-   [E38N+S40T+S99T+R137P+R139P+S142P]huIFNG,-   [E38N+S40T+S99T+R137P+R140P+S142P]huIFNG,-   [E38N+S40T+S99T+R139P+R140P+S142P]huIFNG and-   [E38N+S40T+S99T+R137P+R139P+R140P+S142P]huIFNG,-   more preferably selected from the group consisting of-   [E38N+S40T+S99T+S132P+R137P+RI40P]huIFNG,-   [E38N+S40T+S99T+S132P+R140P)huIFNG,-   [E38N+S40T+S99T+R137P+R139P+S142P]huIFNG and-   [E38N+S40T+S99T+R137P+S142P]huIFNG,-   most preferably selected from the group consisting of-   [E38N+S40T+S99T+S132P+R137P+R140P]huIFNG and-   [E38N+S40T+S99T+R137P+R139P+S142P]huIFNG.

Other specific examples of interesting full-length variants of theinvention include variants selected from the group consisting of

-   [G18T+R137P+R140P]huIFNG,-   [G18T+S99T+R137P+R140P]huIFNG,-   [E38N+S40T+R137P+R140P]huIFNG,-   [E38N+S40T+S99T+R137P+R140P]huIFNG,-   [K61T+R137P+R140P]huIFNG,-   [K61T+S99T+R137P+R140P]huIFNG,-   [S65N+Q67T+R137P+R140P]huIFNG,-   [S65N+Q67T+S99T+R137P+R140P]huIFNG,-   [N85T+R137P+R140P]huIFNG and-   [N85T+S99T+R137P+R140P]huIFNG,-   more preferably selected from the group consisting of-   [G18T+S99T+R137P+R140P]huIFNG,-   [E38N+S40T+S99T+R137P+R140P]huIFNG,-   [K61T+S99T+R137P+R140P]huIFNG,-   [S65N+Q67T+S99T+R137P+R140P]huIFNG, and-   [N85T+S99T+R137P+R140P]huIFNG,-   most preferably [E38N+S40T+S99T+R137P+R140P]huIFNG.

The IFNG polypeptide variant of the invention preferably contains asingle additional in vivo glycosylation site in the amino acid sequencefrom residue no. 1 to residue no. 131. However, in order to become of asufficient size to increase the functional in vivo half-life or theserum half-life it may be desirable that the polypeptide variantcomprises more than one additional in vivo N-glycosylation site, inparticular 2-7 or 2-5 additional in vivo N-glycosylation sites, such as2, 3, 4, 5, 6 or 7 in vivo N-glycosylation sites. Such in vivoN-glycosylation sites are preferably introduced by one or moresubstitutions described in any of the above lists.

Thus, in another interesting embodiment of the invention, the IFNGpolypeptide comprises at least two introduced glycosylation sites, inparticular at least two introduced N-glycosylation sites in the aminoacid sequence from residue no. 1 to residue no. 131.

The at least two modifications, in particular substitutions, leading tothe introduction of the at least two introduced N-glycosylation sitesmay preferably be selected from the group consisting of G18S, G18T,E38N, E38N+S40T, K61S, K61T, S65N+Q67S, S65N+Q67T, N85S, N85T, K94N,Q106S and Q106T, more preferably selected from the group consisting ofG18T, E38N+S40T, K61T, S65N+Q67T, N85T, K94N and Q106T, even morepreferably selected from the group consisting of G18T, E38N+S40T, K61T,S65N+Q67T and N85T.

Specific examples of such substitutions giving rise to an IFNG variantcomprising at least two additional N-glycosylation sites in the aminoacid sequence from residue no. 1 to residue no. 131 include:G18T+E38N+S40T, G18T+K61T, G18T+S65N+Q67T, G18T+N85T, E38N+S40T+K61T,E38N+S40T+S65N+Q67T, E38N+S40T+N85T, K61T+S65N+Q67T, K61T+N85T andS65N+Q67T+N85T.

Preferably, any of the above-mentioned modified IFNG variants furthercomprises the substitution S99T.

Furthermore, the naturally occurring N-glycosylation site located atposition 25 may be removed. This may be done by removing the N25 residueand/or by removing the T27 residue, preferably by substitution.Preferably, the N-glycosylation site located at position 25 may beremoved by the substitution N25G+T27P.

It will be understood that any of the above-mentioned modifications maybe combined with any of the modifications disclosed in the sectionentitled “IFNG variants with optimised in vivo glycosylation sites”, inparticular with the substitution S99T and/or with any of themodifications disclosed in the section entitled “IFNG variants whereinthe non-polypeptide moiety is a molecule, which has cysteine as anattachment group”.

IFNG Variants Wherein the Non-Polypeptide Moiety is a Molecule, Whichhas Cysteine as an Attachment Group

In another particular preferred embodiment of the invention the IFNGvariant comprises, in the amino acid sequence from residue no. 1 toresidue no. 131, at least one introduced cysteine residue. Preferably,the cysteine residue is introduced by substitution.

For instance, a cysteine residue may be introduced into a position ofthe IFNG polypeptide (from residue no. 1 to residue no. 131), whichcomprises an amino acid residue exposed to the surface. Preferably saidsurface-exposed amino acid residue has at least 25% of the side chainexposed to the surface, in particular at least 50% of its side chainexposed to the surface. Details regarding determination of suchpositions can be found in Example 1 herein.

For instance, substitutions that lead to introduction of a cysteineresidue at positions exposed at the surface of the IFNG polypeptide andoccupied by amino acid residue having at least 25% of the side chainexposed to the surface (in a structure with receptor molecule) include:Q1C, D2C, P3C, K6C, E9C, N10C, K13C, Y14C, N16C, G18C, H19C, D21C, N25C,G26C, G31C, K34C, N35C, K37C, E38C, E39C, S40C, K55C, K58C, N59C, K61C,D62C, D63C, Q64C, S65C, Q67C, K68C, E71C, T72C, K74C, E75C, N78C, V79C,K80C, N83C, S84C, N85C, K86C, K87C, D90C, E93C, K94C, T101C, D102C,L103C, N104C and E119C.

Substitutions that lead to introduction of a cysteine residue atpositions exposed at the surface of the IFNG polypeptide and occupied byamino acid residue having at least 50% of the side chain exposed to thesurface (in a structure with receptor molecule) include: P3C, K6C, N10C,K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C,N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C,T101C, D102C, L103C and N104C.

Usually, it is not preferred to introduce cysteine residue (andsubsequently attaching these cysteine residue to a non-polypeptidemoiety) in the region constituting the receptor binding site (except inspecial cases, cf. the section entitled “Variants with a reducedreceptor affinity”). Accordingly, the mutations Q1C, E9C, G18C, H19C,D21C, G26C, K34C, K37C and E119C should normally not be performed,unless a reduced receptor affinity is desired. In addition, the positivecluster K128, R129, K130 and R131 is required for activity and shouldnormally not be modified.

Most preferably, said cysteine residue is introduced by a substitutionselected from the group consisting of N10C, N16C, E38C, N59C, S65C,N83C, K94C, N104C and A124C, such as N16C, N59C and N16C+N59C. In ahigly preferred embodiment of the invention said cysteine residue isintroduced by the substitution N16C or N59C.

Thus, specific examples of interesting full-length variants of theinvention include variants selected from the group consisting of

-   [N16C+S99T+S132P+R137P]huIFNG,-   [N16C+S99T+S132P+R139P]huIFNG,-   [N16C+S99T+S132P+R140P]huIFNG,-   [N16C+S99T+S132P+R137P+R139P]huIFNG,-   [N16C+S99T+S132P+R137P+R140P]huIFNG,-   [N16C+S99T+S132P+R139P+R140P]huIFNG,-   [N16C+S99T+S132P+R137P+R139P+R140P]huIFNG,-   [N59C+S99T+S132P+R137P]huIFNG,-   [N59C+S99T+S132P+R139P]huIFNG,-   [N59C+S99T+S132P+R140P]huIFNG,-   [N59C+S99T+S132P+R137P+R139P]huIFNG,-   [N59C+S99T+S132P+R137P+R140P]huIFNG,-   [N59C+S99T+S132P+R139P+R140P]huIFNG,-   [N59C+S99T+S132P+R137P+R139P+R140P]huIFNG,-   [N16C+S99T+R137P+S142P]huIFNG,-   [N16C+S99T+R139P+S142P]huIFNG,-   [N16C+S99T+R140P+S142P]huIFNG,-   [N16C+S99T+R137P+R139P+S142P]huIFNG,-   [N16C+S99T+R137P+R140P+S142P]huIFNG,-   [N16C+S99T+R139P+R140P+S142P]huIFNG,-   [N16C+S99T+R137P+R139P+R140P+S142P]huIFNG,-   [N59C+S99T+R137P+S142P]huIFNG,-   [N59C+S99T+R139P+S142P]huIFNG,-   [N59C+S99T+R140P+S142P]huIFNG,-   [N59C+S99T+R137P+R139P+S142P]huIFNG,-   [N59C+S99T+R137P+R140P+S142P]huIFNG,-   [N59C+S99T+R139P+R140P+S142P]huIFNG and-   [N59C+S99T+R137P+R139P+R140P+S142P]huIFNG,-   more preferably selected from the group consisting of-   [N16C+S99T+S132P+R137P+R140P]huIFNG,-   [N16C+S99T+S132P+R140P]huIFNG,-   [N59C+S99T+S132P+R137P+R140P]huIFNG,-   [N59C+S99T+S132P+R140P]huIFNG,-   [N16C+S99T+R137P+R139P+S142P]huIFNG,-   [N16C+S99T+R137P+S142P]huIFNG,-   [N59C+S99T+R137P+R139P+S142P]huIFNG and-   [N59C+S99T+R137P+S142P]huIFNG,-   most preferably selected from the group consisting of-   [N16C+S99T+S132P+R137P+R140P]huIFNG,-   [N59C+S99T+S132P+R137P+R140P]huIFNG,-   [N16C+S99T+R137P+R139P+S142P]huIFNG and-   [N59C+S99T+R137P+R139P+S142P]huIFNG.

Other specific examples of interesting full-length variants of theinvention include variants selected from the group consisting of

-   [N10C+R137P+R140P]huIFNG,-   [N10C+R137P+R140P]Met-huIFNG,-   [N10C+S99T+R137P+R140P]huIFNG,-   [N16C+R137P+R140P]huIFNG,-   [N16C+R137P+R140P]Met-huIFNG,-   [N16C+S99T+R137P+R140P]huIFNG,-   [E38C+R137P+R140P]huIFNG,-   [E38C+R137P+R140P]Met-huIFNG,-   [E38C+S99T+R137P+R140P]huIFNG,-   [N59C+R137P+R140P]huIFNG,-   [N59C+R137P+R140P]Met-huIFNG,-   [N59C+S99T+R137P+R140P]huIFNG,-   [S65C+R137P+R140P]huIFNG,-   [S65C+R137P+R140P]Met-huIFNG,-   [S65C+S99T+R137P+R140P]huIFNG,-   [N83C+R137P+R140P]huIFNG,-   [N83C+R137P+R140P]Met-huIFNG,-   [N83C+S99T+R137P+R140P]huIFNG,-   [K94C+R137P+R140P]huIFNG,-   [K94C+R137P+R140P]Met-huIFNG,-   [K94C+S99T+R137P+R140P]huIFNG,-   [N104C+R137P+R140P]huIFNG,-   [N104C+R137P+R140P]Met-huIFNG,-   [N104C+S99T+R137P+R140P]huIFNG,-   [A124C+R137P+R140P]huIFNG,-   [A124C+R137P+R140P]Met-huIFNG and-   [A124C+S99T+R137P+R140P]huIFNG,-   more preferably selected from the group consisting of-   [N16C+R137P+R140P]huIFNG,-   [N16C+R137P+R140P]Met-huIFNG,-   [N16C+S99T+R137P+R140P]huIFNG,-   [N59C+R137P+R140P]huIFNG,-   [N59C+R137P+R140P]Met-huIFNG and-   [N59C+S99T+R137P+R140P]huIFNG,-   even more preferably selected from the group consisting of-   [N16C+R137P+R140P]huIFNG,-   [N16C+S99T+R137P+R140P]huIFNG,-   [N59C+R137P+R140P]huIFNG and-   [N59C+S99T+R137P+R140P]huIFNG,-   most preferably selected from the group consisting of-   [N16C+S99T+R137P+R140P]huIFNG and-   [N59C+S99T+R137P+R140P]huIFNG.

The IFNG variant of the invention preferably contains a single cysteineresidue in the amino acid sequence from residue no. 1 to residue no.131. However, in order to become of a sufficient size to increase thefunctional in vivo half-life or the serum half-life it may be desirablethat the polypeptide comprises more than one cysteine, in particular 2-7or 2-5 cysteine residues, such as 2, 3, 4, 5, 6 or 7 cysteine residues.Such cysteine residues are preferably introduced by one or moresubstitutions described in any of the above lists.

Thus, in another embodiment of the invention, the IFNG variant comprisesat least two introduced cysteine residues in the amino acid sequencefrom residue no. 1 to residue no. 131.

The at least two modifications, in particular substitutions, leading tothe introduction of the at least two cysteine residues may preferably beselected from the group consisting of N10C, N16C, E38C, N59C, S65C,N83C, K94C, N104C and A124C. Specific examples of such substitutionsgiving rise to an IFNG polypeptide comprising at least two cysteineresidues (in the amino acid sequence from residue no.1 to residue no.131) include: N10C+N16C, N10C+E38C, N10C+N59C, N10C+S65C, N10C+N83C,N10C+K94C, N10C+N104C, N10C+A124C, N16C+E38C, N16C+N59C, N16C+S65C,N16C+N83C, N16C+K94C, N16C+N104C, N16C+A124C, E38C+N59C, E38C+S65C,E38C+N83C, E38C+K94C, E38C+N104C, E38C+A124C, N59C+S65C, N59C+N83C,N59C+K94C, N59C+N104C, N59C+A124C, S65C+N83C, S65C+K94C, S65C+N104C,S65C+A124C, N83C+K94C, N83C+N104C, N83C+A124C, K94C+N104C, K94C+A124Cand N104C+A124C, in particular N16C+N59C.

Preferably, any of the above-mentioned modified IFNG polypeptidesfurther comprises the substitution S99T.

Furthermore, the naturally occurring N-glycosylation site located atposition 25 may be removed. This may be done by removing the N25 residueand/or by removing the T27 residue, preferably by substitution.Preferably, the N-glycosylation site located at position 25 may beremoved by the substitution N25G+T27P.

As will be understood the introduced cysteine residue(s) may preferablybe conjugated to a non-polypeptide moiety, such as PEG or morepreferably mPEG. The conjugation between the cysteine-containingpolypeptide and the polymer molecule may be achieved in any suitablemanner, e.g. as described in the section entitled “Conjugation to apolymer molecule”, e.g. in using a one step method or in the stepwisemanner referred to in said section. The preferred method for PEGylatingthe IFNG polypeptide is to covalently attach PEG to cysteine residuesusing cysteine-reactive PEGs. A number of highly specific,cysteine-reactive PEGs with different groups (e.g. maleimide,vinylsulfone and orthopyridyl-disulfide) and different size PEGs (2-20kDa, such as 5 kDa, 10 kDa, 12 kDa or 15 kDa) are commerciallyavailable, e.g. from Shearwater Polymers Inc., Huntsville, Ala., USA).

It will be understood that any of the above-mentioned modifications maybe combined with any of the modifications disclosed in the sectionentitled “IFNG variants with optimised in vivo glycosylation sites”, inparticular with the substitution S99T and/or with any of themodifications disclosed in the section entitled “IFNG variants whereinthe non-polypeptide moiety is a sugar moiety”.

IFNG Variants Wherein a First Non-Polypeptide Moiety is a Sugar Moietyand a Second Non-Polypeptide Moiety is a Molecule, Which has Cysteine asan Attachment Group

In a further embodiment of the invention the IFNG variant comprises, inthe amino acid sequence from residue no. 1 to residue no. 131, at leastone introduced N-glycosylation site and at least one introduced cysteineresidue. Preferably, the cysteine residue and the N-glycosylation siteis introduced by substitution. Such polypeptides may be prepared byselecting the residues described in the two preceding sectionsdescribing suitable positions for introducing N-glycosylation sites andcysteine residues, respectively. However, in a preferred embodiment ofthe invention said IFNG variants comprises substitutions selected fromthe group consisting of G18T+N10C, G18T+E38C, G18T+N59C, G18T+S65C,G18T+N83C, G18T+K94C, G18T+N104C, G18T+A124C, E38N+S40T+N10C,E38N+S40T+K94C, E38N+S40T+N59C, E38N+S40T+S65C, E38N+S40T+N83C,E38N+S40T+K94C, E38N+S40T+N104C, E38N+S40T+A124C, K61T+N10C, K61T+N16C,K61T+E38C, K61T+S65C, K61T+N83C, K61T+K94C, K61T+N104C, K61T+A124C,N85T+N10C, S65N+Q67T+N10C, S65N+Q67T+N16C, S65N+Q67T+E38C,S65N+Q67T+N59C, S65N+Q67T+N83C, S65N+Q67T+K94C, S65N+Q67T+N104C,S65N+Q67T+A124C, N85T+N10C, N85T+N16C, N85T+E38C, N85T+N59C, N85T+S65C,N85T+K94C, N85T+N104C, N95T+A124C, K94N+N10C, K94N+N16C, K94N+E38C,K94N+N59C, K94N+S65C, K94N+N83C, K94N+N104C, K94N+A124C, Q106T+N10C,Q106T+N16C, Q106T+E38C, Q106T+N59C, Q106T+S65T, Q106T+N83C, Q106T+K94Cand Q106T+A124C, more preferably selected from the group consisting ofE38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C, E38N+S40T+S65C,E38N+S40T+N83C, E38N+S40T+K94C, E38N+S40T+N104C and E38N+S40T+A124C,most preferably selected from the group consisting of E38N+S40T+N16C andE38N+S40T+N59C.

Preferably, any of the above-mentioned modified IFNG polypeptidesfurther comprises the substitution S99T.

In a still further interesting embodiment of the invention the IFNGvariant comprises, in the amino acid sequence from residue no. 1 toresidue no. 131, at least one removed N-glycosylation site and at leastone introduced cysteine residue. Preferably, the cysteine residue isintroduced by substitution and the N-glycosylation site is removed bysubstitution.

In particular, the removed N-glycosylation site is the N-glycosylationsite located at position 25. This may be done by removing the N25residue and/or by removing the T27 residue, preferably by thesubstitution N25G+T27P.

It will be understood that any of the above-mentioned modifications maybe combined with any of the modifications disclosed in the sectionentitled “IFNG variants with optimised in vivo glycosylation sites”, inparticular with the substitution S99T.

IFNG Variants with a Reduced Receptor Affinity

One way to increase the serum half-life or the functional in vivohalf-life of an IFNG polypeptide would be to decrease thereceptor-mediated internalisation and thereby decrease thereceptor-mediated clearance. The receptor-mediated internalisation isdependent upon the affinity of the IFNG dimer for the IFNG receptorcomplex and, accordingly, an IFNG polypeptide with a decreased affinityto the IFNG receptor complex is expected to be internalised, and hencecleared, to a lesser extent.

The affinity of the IFNG dimer to its receptor complex may be decreasedby performing one or more modifications, in particular substitutions, inthe recpetor binding region of the IFNG polypeptide. The amino acidresidues which constitute the receptor binding region is defined inExample 2 herein. One class of substitutions that may be performed isconservative amino acid substitutions. In another embodiment, themodification performed gives rise to the introduction of anN-glycosylation site.

Thus, in a further interesting embodiment of the invention the IFNGpolypeptide comprises, in the amino acid sequence from residue no. 1 toresidue no. 131, at least one modification in the receptor binding site(as defined herein). More particularly, the IFNG polypeptide comprisesat least one substitution, preferably a substitution, which creates anin vivo N-glycosylation site, in said receptor binding region. Forinstance, such substitutions may be selected from the group consistingof Q1N+P3S/T, D2N+Y4S/T, Y4N+K6S/T, V5N+E7S/T, E9N+L11S/T, K12N+Y14S/T,G18N, G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, V22N+D24S/T,D24N+G26S/T, G26N+L28S/T, L30N+I32S/T, K34N+W36S/T, K37N+E39S/T,K108N+I110S/T, H111N+L113S/T, E112N+I114S/T, I114N+V116S/T,Q115N+M117S/T, A118N+L120S/T, E119N and E119N+S121T, preferably from thegroup consisting of Q1N+P3S/T, D2N+Y4S/T, E9N+L11S/T, K12N+Y14S/T, G18N,G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, K34N+W36S/T,K37N+E39S/T, H111N+L113S/T, Q115N+M117S/T, A118N+L120S/T, E119N andE119N+S121T (introduction of N-glycosylation sites in positionscomprising an amino acid residue having at least 25% of its side chainexposed to the surface), more preferably from the group consisting ofQ1N+P3S/T, D2N+Y4S/T, E9N+L11S/T, G18N, G18N+S20T, H19N+D21S/T,S20N+V22S/T, D21N+A23S/T, K34N+W36S/T, K37N+E39S/T, Q115N+M117S/T,A118N+L120S/T, E119N and E119N+S121T (introduction of N-glycosylationsites in positions comprising an amino acid residue having at least 50%of its side chain exposed to the surface), even more preferably from thegroup consisting of Q1N+P3T, D2N+Y4T, E9N+L11T, G18N+S20T, H19N+D21T,S20N+V22T, D21N+A23T, K34N+W36T, K37N+E39T, Q115N+M117T, A118N+L120T andE119N+S121T, most preferably from the group consisting of G18N+S20T,H19N+D21T, D21N+A23T and E119N+S121T, in particular D21N+A23T.

Such variants are contemplated to exhibit a reduced receptor affinity ascompared to glycosylated huIFNG, glycosylated [S99T]huIFNG orActimmune®. The receptor affinity may be measured by any suitable assayand will be known to the person skilled in the art. One example of asuitable assay for determining the receptor binding affinity is theBIAcore® assay described in Michiels et al. Int. J. Biochem. Cell Biol.30:505-516 (1998). Using the above-identified assay, IFNG polypeptidesconsidered useful for the purposes described herein are such IFNGpolypeptides, wherein the binding affinity (K_(d)) is 1-95% of theK_(d)-value of glycosylated huIFNG, glycosylated [S99T]huIFNG orActimmune®. For example the K_(d)-value of the IFNG polypeptide may be1-75% or 1-50%, such as 1-25%, e.g. 1-20% or even as low as 1-15%, 1-10%or 1-5% of the K_(d)-value of glycosylated huIFNG, glycosylated[S99T]huIFNG or Actmmune®.

Typically, such IFNG polypeptides having reduced receptor affinity willexhibit a reduced IFNG activity, e.g. when tested in the “Primary Assay”described herein. For example, the IFNG polypeptide may exhibit 1-95%(e.g. 5-95%) of the IFNG activity of glycosylated huIFNG, glycosylated[S99T]huIFNG or Actimmune®, e.g. 1-75% (e.g. 5-75%), such as 1-50% (e.g.5-50%), e.g. 1-20% (e.g. 5-20%) or 1-10% (e.g. 5-10%) of the IFNG aglycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®D.

As mentioned above, such IFNG polypeptides are contemplated to possessan increased half-life due to the reduced receptor-mediated clearance.Therefore, the IFNG polypeptides according to this aspect of theinvention are contemplated to fulfil the requirements with respect toincreased half-life described previously herein in connection with thedefinition of increased half-life.

Furthermore, it is contemplated that at least some of the cysteinevariant, wherein said cysteine is covalently attached to anon-polypeptide moiety, such as PEG, also exhibit a reducedreceptor-binding affinity and hence a lowered IFNG activity when testedin the “Primary Assay” described herein. It is envisaged that thisproperty may be achieved independently of whether the cysteine (andhence the non-polypeptide moiety) is introduced in the receptor bindingsite since the non-polypeptide moiety is normally of such size that itmay interact/partially impair binding to the IFNG polypeptide to itsreceptor independently of whether said moiety is introduced in thereceptor binding site or not.

Evidently, any of the above-mentioned modifications giving rise to areduced receptor binding affinity may be combined with any of the othermodifications disclosed herein, in particular the modificationsmentioned in the sections entitled “IFNG variants with optimisedN-glycosylation sites”, “IFNG variants wherein the non-polypeptidemoiety is a sugar moiety”, “IFNG variants wherein the non-polypeptidemoiety is a molecule, which has cysteine as an attachment group” and“IFNG variants wherein the first non-polypeptide moiety is a sugarmoiety and the second non-polypeptide moiety is a molecule, which hascysteine as an attachment group”, such as the modifications selectedfrom the group consisiting of E38N+S40T, S99T and combinations thereof.

Analysis of Truncation of IFNG Variants

Determination of C-terminal truncation of purified samples of IFNGpolypeptides can be carried out in a number of ways.

One way of elucidating C-terminal truncations of IFNG polypeptidesrelies on accurate mass determinations by mass spectrometry.Unfortunately, the glycosylation of IFNG is heterogeneous thus making itextremely difficult to determine an accurate mass directly on theglycoprotein. Therefore, different levels of enzymatic deglycosylationare typically used in combination with mass spectrometry.

In one method, the entire glycan part of the IFNG polypeptide is cleavedof using the endo-glycosidase PNGase F followed by accurate massdetermination using either ESI mass spectrometry or MALDI-TOF massspectrometry. Comparing the experimental masses to the known amino acidsequence of IFNG makes it possible to determine the sites of C-terminaltruncation.

In another related method, only the sialic acid of the glycan part ofthe IFNG polypeptide is cleaved off instead of the entire glycan. Insome cases this is sufficient to reduce the heterogeneity of the sampleto a level where the sites of C-terminal truncations can be deducedfollowing accurate mass determination using either ESI mass spectrometryor MALDI-TOF mass spectrometry.

A more traditional way of elucidating C-terminal truncations of IFNGpolypeptides employs peptide mapping in combination with massspectrometry and chemical amino acid sequencing. In brief, the IFNGpolypeptide is degraded with a protease of known specificity (e.g. Asp-Nprotease) followed by peptide separation using RP-HPLC. Fractions canthen by mass analysed either on-line using ESI mass spectrometry oroff-line using MALDI-TOF mass spectrometry. Comparing the massesobtained for peptides with the known amino acid sequence of IFNG makesit possible to determine the likely sites of C-terminal truncation.Verification can then be obtained through amino acid sequencing.

Conjugation Methods

The Non-Polypeptide Moiety

As indicated further above the non-polypeptide moiety is preferablyselected from the group consisting of a sugar moiety (e.g. by way of invivo N-glycosylation), a polymer molecule, a lipophilic compound and anorganic derivatizing agent. All of these agents may confer desirableproperties to the IFNG polypeptide, in particular increased AUC_(sc),increased serum half-life, increased functional in vivo half-life whenadministered intravenously, reduced immunogenicity and/or increasedbioavailability. The polypeptide is normally conjugated to only one typeof non-polypeptide moiety, but may also be conjugated to two or moredifferent types of non-polypeptide moieties, e.g. to a polymer moleculeand a sugar moiety, to a lipophilic group and a sugar moiety, to anorganic derivating agent and a sugar moiety, to a lipophilic group and apolymer molecule, etc. When conjugated to two different types ofnon-polypeptide moieties these are preferably a sugar moiety and apolymer moiety. The conjugation to two or more different non-polypeptidemoieties may be done simultaneous or sequentially. In the followingsections “Conjugation to a lipophilic compound”, “Conjugation to apolymer molecule”, “Conjugation to a sugar moiety” and “Conjugation toan organic derivatizing agent” conjugation to specific types ofnon-polypeptide moieties is described.

Conjugation to a Lipophilic Compound

The polypeptide and the lipophilic compound may be conjugated to eachother, either directly or by use of a linker. The lipophilic compoundmay be a natural compound such as a saturated or unsaturated fatty acid,a fatty acid diketone, a terpene, a prostaglandin, a vitamine, acarotenoide or steroide, or a synthetic compound such as a carbon acid,an alcohol, an amine and sulphonic acid with one or more alkyl-, aryl-,alkenyl- or other multiple unsaturated compounds. The conjugationbetween the polypeptide and the lipophilic compound, optionally througha linker may be done according to methods known in the art, e.g. asdescribed by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976and in WO 96/12505.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range of300-100,000 Da or 1000-50,000 Da, such as in the range of 2000-40,000 Daor 2000-30,000 Da, e.g. in the range of 2000-20,000 Da, 2000-10,000 Daor 1000-5000 Da. More specifically, the polymer molecule, such as PEG,in particular mPEG, will typically have a molecular weight of about 2,5, 10, 12, 15, 20, 30, 40 or 50 kDa.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer, which comprises one or more differentcoupling groups, such as, e.g., a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life and/or increasing the AUC_(sc). Another example of apolymer molecule is human albumin or another abundant plasma protein.Generally, polyalkylene glycol-derived polymers are biocompatible,non-toxic, non-antigenic, non-immunogenic, have various water solubilityproperties, and are easily secreted from living organisms.

PEG is the preferred polymer molecule to be used, since it has only fewreactive groups capable of cross-linking compared, e.g., topolysaccharides such as dextran, and the like. In particular,monofunctional PEG, e.g., monomethoxypolyethylene glycol (mPEG), is ofinterest since its coupling chemistry is relatively simple (only onereactive group is available for conjugating with attachment groups onthe polypeptide). Consequently, the risk of cross-linking is eliminated,the resulting conjugated polypeptides are more homogeneous and thereaction of the polymer molecules with the polypeptide is easier tocontrol.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate(SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitablyactivated polymer molecules are commercially available, e.g. fromShearwater Polymers, Inc., Huntsville, Ala., USA. Alternatively, thepolymer molecules can be activated by conventional methods known in theart, e.g. as disclosed in WO 90/13540. Specific examples of activatedlinear or branched polymer molecules for use in the present inventionare described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogue(Functionalized Biocompatible Polymers for Research and pharmaceuticals,Polyethylene Glycol and Derivatives, incorporated herein by reference).Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, and SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, AID-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 andU.S. Pat. No. 5,643,575, both of which references are incorporatedherein by reference. Furthermore, the following publications,incorporated herein by reference, disclose useful polymer moleculesand/or PEGylation chemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No.5,476,653, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502,U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No.5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No.5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat.No. 5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400472, EP 183 503 and EP 154 316.

The conjugation of the polypeptide variant and the activated polymermolecules is conducted by use of any conventional method, e.g. asdescribed in the following references (which also describe suitablemethods for activation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.).

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe variant polypeptide (examples of which are given further above), aswell as the functional groups of the polymer (e.g. being amine,hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide,vinysulfone or haloacetate). The PEGylation may be directed towardsconjugation to all available attachment groups on the variantpolypeptide (i.e. such attachment groups that are exposed at the surfaceof the polypeptide) or may be directed towards one or more specificattachment groups, e.g. the N-terminal amino group as described in U.S.Pat. No. 5,985,265 or to cysteine residues. Furthermore, the conjugationmay be achieved in one step or in a stepwise manner (e.g. as describedin WO 99/55377).

In a particular interesting embodiment PEGylation is achieved byconjugatin the PEG group(s) to introduced cysteine residues. Specificexamples of activated PEG polymers particularly preferred for couplingto cysteine residues, include the following linear PEGs:vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG);maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) andorthopyridyl-disulfide-PEG (OPSS-PEG), preferablyorthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEGpolymers will have a size of about 5 kDa, about 10 kD, about 12 kDa orabout 20 kDa.

For PEGylation to cysteine residues the IFNG variant is usually treatedwith a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.The reducing agent is subsequently removed by any conventional method,such as by desalting. Conjugation of PEG to a cysteine residue typicallytakes place in a suitable buffer at pH 6-9 at temperatures varying from4° C. to 25° C. for periods up to 16 hours.

It will be understood, that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form (e.g. whether they are linear or branched)of such molecules, and where in the polypeptide such molecules areattached. For instance, the molecular weight of the polymer to be usedmay be chosen on the basis of the desired effect to be achieved. Forinstance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight (e.g. to reduce renalclearance) it may be desirable to conjugate as few high Mw polymermolecules as possible to obtain the desired molecular weight. When ahigh degree of epitope shielding is desirable this may be obtained byuse of a sufficiently high number of low molecular weight polymer (e.g.with a molecular weight of about 5,000 Da) to effectively shield all ormost epitopes of the polypeptide. For instance, 2-8, such as 3-6 suchpolymers may be used.

In connection with conjugation to only a single attachment group on theprotein (as described in U.S. Pat. No. 5,985,265), it may beadvantageous that the polymer molecule, which may be linear or branched,has a high molecular weight, e.g. about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimingat reacting all available polymer attachment groups with polymermolecules. Typically, the molar ratio of activated polymer molecules topolypeptide is 1000-1, in particular 200-1, preferably 100-1, such as10-1 or 5-1 in order to obtain optimal reaction. However, also equimolarratios may be used.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378.

Subsequent to the conjugation residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules removed by a suitable method.

Coupling to a Sugar Moiety

The coupling of a sugar moiety may take place in vivo or in vitro. Inorder to achieve in vivo glycosylation of a polypeptide with IFNGactivity, which have been modified so as to introduce one or more invivo glycosylation sites (see the section “IFNG variants wherein thenon-polypeptide moiety is a sugar moiety), the nucleotide sequenceencoding the polypeptide variant must be inserted in a glycosylating,eukaryotic expression host. The expression host cell may be selectedfrom fungal (filamentous fungal or yeast), insect or animal cells orfrom transgenic plant cells. Furthermore, the glycosylation may beachieved in the human body when using a nucleotide sequence encoding thepolypeptide of the invention in gene therapy. In one embodiment the hostcell is a mammalian cell, such as a CHO cell, a BHK cell or a HEK cell,e.g. a HEK293 cell, or an insect cell, such as an SF9 cell, or a yeastcell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any othersuitable glycosylating host, e.g. as described further below.Optionally, sugar moieties attached to the IFNG polypeptide by in vivoglycosylation are further modified by use of glycosyltransferases, e.g.using the glycoAdvance™ technology marketed by Neose, Horsham, Pa., USA.Thereby, it is possible to, e.g., increase the sialyation of theglycosylated IFNG polypeptide following expression and in vivoglycosylation by CHO cells.

Covalent in vitro coupling of glycosides to amino acid residues of IFNGmay be used to modify or increase the number or profile of carbohydratesubstituents. Depending on the coupling mode used, the sugar(s) may beattached to a) arginine and histidine, b) free carboxyl groups, c) freesulfhydryl groups such as those of cysteine, d) free hydroxyl groupssuch as those of serine, threonine, tyrosine or hydroxyproline, e)aromatic residues such as those of phenylalanine or tryptophan or f) theamide group of glutamine. These amino acid residues constitute examplesof attachment groups for a sugar moiety, which may be introduced and/orremoved in the IFNG polypeptide. Suitable methods of in vitro couplingare described, for example, in WO 87105330 and in Aplin et al., CRC CritRev. Biochem., pp. 259-306, 1981. The in vitro coupling of sugarmoieties or PEG to protein- and peptide-bound Gln-residues can also becarried out by transglutaminases (TGases), e.g. as described by Sato etal., 1996 Biochemistry 35, 13072-13080 or in EP 725145.

Coupling to an Organic Derivatizing Agent

Covalent modification of the IFNG polypeptide may be performed byreacting (an) attachment group(s) of the polypeptide with an organicderivatizing agent. Suitable derivatizing agents and methods are wellknown in the art. For example, cysteinyl residues most commonly arereacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino terminal residues are reacted with succinicor other carboxylic acid anhydrides. Derivatization with these agentshas the effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues are modified by reaction with one orseveral conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of Functional Site

It has been reported that excessive polymer conjugation can lead to aloss of activity of the polypeptide to which the polymer is conjugated.This problem can be eliminated, e.g., by removal of attachment groupslocated at the functional site or by blocking the functional site priorto conjugation. These latter strategies constitute further embodimentsof the invention, the first strategy being exemplified further above,e.g. by removal of lysine residues which may be located close to thefunctional site and/or by introducing cysteine residues and/or in vivoglycosylation sites at positions not interfering with functional sites.

More specifically, according to the latter strategy the conjugationbetween the polypeptide and the non-polypeptide moiety is conductedunder conditions where the functional site of the IFNG polypeptide isblocked by a helper molecule capable of binding to the functional siteof the polypeptide. Preferably, the helper molecule is one, whichspecifically recognizes a functional site of the polypeptide, such as areceptor. Alternatively, the helper molecule may be an antibody, inparticular a monoclonal antibody recognizing the polypeptide exhibitingIFNG activity. In particular, the helper molecule may be a neutralizingmonoclonal antibody.

The polypeptide is allowed to interact with the helper molecule beforeeffecting conjugation. This ensures that the functional site of thepolypeptide is shielded or protected and consequently unavailable forderivatization by the non-polypeptide moiety such, as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, a sugar moiety, anorganic derivatizing agent or any other compound is conducted in thenormal way, e.g. as described in the sections above entitled“Conjugation to . . . ” and “Coupling to . . . ”.

In a further embodiment the helper molecule is first covalently linkedto a solid phase such as column packing materials, for instance Sephadexor agarose beads, or a surface, e.g. reaction vessel. Subsequently, thepolypeptide is loaded onto the column material carrying the helpermolecule and conjugation carried out according to methods known in theart, e.g. as described in the sections above entitled “Conjugation to .. . ” and “Coupling to . . . ”. This procedure allows the conjugatedpolypeptide to be separated from the helper molecule by elution. Theconjugated polypeptide is eluted by conventional techniques underphysico-chemical conditions that do not lead to a substantivedegradation of the conjugated polypeptide. The fluid phase containingthe conjugated polypeptide is separated from the solid phase to whichthe helper molecule remains covalently linked. The separation can beachieved in other ways: For instance, the helper molecule may bederivatized with a second molecule (e.g. biotin) that can be recognizedby a specific binder (e.g. streptavidin). The specific binder may belinked to a solid phase thereby allowing the separation of theconjugated polypeptide from the helper molecule-second molecule complexthrough passage over a second helper-solid phase column which willretain, upon subsequent elution, the helper molecule-second moleculecomplex, but not the polypeptide conjugate. The conjugated polypeptidemay be released from the helper molecule in any appropriate fashion.De-protection may be achieved by providing conditions in which thehelper molecule dissociates from the functional site of the IFNG towhich it is bound. For instance, a complex between an antibody to whicha polymer is conjugated and an anti-idiotypic antibody can bedissociated by adjusting the pH to an acid or alkaline pH.

Methods of Preparing Interferon Gamma Variants of the Invention

The IFNG polypeptide variant, preferably in glycosylated form, may beproduced by any suitable method known in the art. Such methods includeconstructing a nucleotide sequence encoding the polypeptide andexpressing the sequence in a suitable transformed or transfected host.However, polypeptides of the invention may be produced, albeit lessefficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

The nucleotide sequence of the invention encoding an IFNG polypeptide(in monomer or single chain form) may be constructed by isolating orsynthesizing a nucleotide sequence encoding the parent IFNG, such ashuIFNG with the amino acid sequence SEQ ID NO:1, and then changing thenucleotide sequence so as to effect introduction (i.e. insertion orsubstitution) or deletion (i.e. removal or substitution) of the relevantamino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with well-known methods, see, e.g., Mark etal., “Site-specific Mutagenesis of the Human Fibroblast InterferonGene”, Proc. Natl. Acad. Sci. USA, 81, pp. 5662-66 (1984); and U.S. Pat.No. 4,588,585.

Alternatively, the nucleotide sequence is prepared by chemicalsynthesis, e.g. by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction (LCR). The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the IFNG in the desired transformed hostcell.

It should of course be understood that not all vectors and expressioncontrol sequences unction equally well to express the nucleotidesequence encoding an IFNG polypeptide described herein. Neither will allhosts function equally well with the same expression system. However,one of skill in the art may make a selection among these vectors,expression control sequences and hosts without undue experimentation.For example, in selecting a vector, the host must be considered becausethe vector must replicate in it or be able to integrate into thechromosome. The vector's copy number, the ability to control that copynumber, and the expression of any other proteins encoded by the vector,such as antibiotic markers, should also e considered. In selecting anexpression control sequence, a variety of factors should also beconsidered. These include, for example, the relative strength of thesequence, its controllability, and its compatibility with the nucleotidesequence encoding the polypeptide, particularly as regards potentialsecondary structures. Hosts should be selected by consideration of theircompatibility with the chosen vector, the toxicity of the product codedfor by the nucleotide sequence, their secretion characteristics, theirability to fold the polypeptide correctly, their fermentation or culturerequirements, and the ease of purification of the products coded for bythe nucleotide sequence.

The recombinant vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotidesequence encoding the IFNG polypeptide is operably linked to additionalsegments required for transcription of the nucleotide sequence. Thevector is typically derived from plasmid or viral DNA. A number ofsuitable expression vectors for expression in the host cells mentionedherein are commercially available or described in the literature. Usefulexpression vectors for eukaryotic hosts, include, for example, vectorscomprising expression control sequences from SV40, bovine papillomavirus, adenovirus and cytomegalovirus. Specific vectors are, e.g.,pCDNA3.1(+)Hyg (Invitrogen, Carlsbad, Calif., USA) and pCI-neo(Stratagene, La Jola, Calif., USA). Useful expression vectors forbacterial hosts include known bacterial plasmids, such as plasmids fromE. coli, including pBR322, pET3a and pET12a (both from Novagen Inc., WI,USA), wider host range plasmids, such as RP4, phage DNAs, e.g., thenumerous derivatives of phage lambda, e.g., NM989, and other DNA phages,such as M13 and filamentous single stranded DNA phages. Usefulexpression vectors for yeast cells include the 2μ plasmid andderivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373), thepJSO37 vector described in (Okkels, Ann. New York Acad. Sci. 782,202-207, 1996) and pPICZ A, B or C Invitrogen). Useful vectors forinsect cells include pVL941, pBG311 (Cate et al., “isolation of theBovine and Human Genes for Mullerian Inhibiting Substance And Expressionof the Human Gene In Animal Cells”, Cell, 45, pp. 685-98 (1986),pBluebac 4.5 and pMelbac (both available from Invitrogen) as well asPVL1392 (available from Pharmingen).

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the IFNG polypeptide to be amplified incopy number. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction Of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication. When the host cell is a yeast cell, suitable sequencesenabling the vector to replicate are the yeast plasmid 2p replicationgenes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. Forfilamentous fungi, selectable markers include amdS pyrG, arcB, niaD, sC.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe IFNG polypeptide. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, enhancer or upstream activatingsequence, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter.

A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, e.g. the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus Elbregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol Aug. 20, 1987; 196 (4):947-50).

In order to improve expression in mammalian cells a synthetic intron maybe inserted in the 5′ untranslated region of the nucleotide sequenceencoding the IFNG polypeptide. An example of a synthetic intron is thesynthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, WI, USA).

Examples of suitable control sequences for directing transcription ininsect cells include the polyhedrin promoter, the P10 promoter, theAutographa californica polyhedrosis virus basic protein promoter, thebaculovirus immediate early gene 1 promoter and the baculovirus 39Kdelayed-early gene promoter, and the SV40 polyadenylation sequence.

Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydogenase genes, the ADH2-4c promoter and theinducible GAL promoter.

Examples of suitable control sequences for use in filamentous fungalhost cells include the ADH3 promoter and terminator, a promoter derivedfrom the genes encoding Aspergillus oryzae TAKA amylase triose phosphateisomerase or alkaline protease, an A. niger α-amylase, A. niger or A.nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor mieheiaspartic proteinase or lipase, the TPI1 terminator and the ADH3terminator.

Examples of suitable control sequences for use in bacterial host cellsinclude promoters of the lac system, the trp system, the TAC or TRCsystem and the major promoter regions of phage lambda.

The nucleotide sequence of the invention, whether prepared bysite-directed mutagenesis, synthesis or other methods, may or may notalso include a nucleotide sequence that encode a signal peptide. Thesignal peptide is present when the polypeptide is to be secreted fromthe cells in which it is expressed. Such signal peptide, if present,should be one recognized by the cell chosen for expression of thepolypeptide. The signal peptide may be homologous (e.g. be that normallyassociated with huIFNG) or heterologous (i.e. originating from anothersource than huIFNG) to the polypeptide or may be homologous orheterologous to the host cell, i.e. be a signal peptide normallyexpressed from the host cell or one which is not normally expressed fromthe host cell. Accordingly, the signal peptide may be prokaryotic, e.g.derived from a bacterium such as E. coli, or eukaryotic, e.g. derivedfrom a mammalian, or insect or yeast cell.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide, theprotein to be expressed (whether it is an intracellular or intracellularprotein) and whether it is desirable to obtain secretion. For use infilamentous fungi, the signal peptide may conveniently be derived from agene encoding an Aspergillus sp. amylase or glucoamylase, a geneencoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosalipase. The signal peptide is preferably derived from a gene encoding A.oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stableamylase, or A. niger glucoamylase. For use in insect cells, the signalpeptide may conveniently be derived from an insect gene (cf. WO90/05783), such as the lepidopteran Manduca sexta adipokinetic hormoneprecursor, (cf. U.S. Pat. No. 5,023,328), the honeybee melittin(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al.,Protein Expression and Purification 4, 349-357 (1993) or humanpancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997).

A preferred signal peptide for use in mammalian cells is that of huIFNGor the murine Ig kappa light chain signal peptide (Coloma, M (1992) J.Imm. Methods 152:89-104). For use in yeast cells suitable signalpeptides have been found to be the α-factor signal peptide from S.cereviciae. (cf. U.S. Pat. No. 4,870,008), the signal peptide of mousesalivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp.643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls etal., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO87/02670), and the yeast aspartic protease 3 (YAP3) signal peptide (cf.M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

Any suitable host may be used to produce the IFNG polypeptide, includingbacteria, fungi (including yeasts), plant, insect, mammal, or otherappropriate animal cells or cell lines, as well as transgenic animals orplants. Examples of bacterial host cells include grampositive bacteriasuch as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonasor Streptomyces, or gramnegative bacteria, such as strains of E. coli.The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

Examples of suitable filamentous fungal host cells include strains ofAspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium orTrichoderma. Fungal cells may be transformed by a process involvingprotoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus host cells are described inEP 238 023 and U.S. Pat. No. 5,679,543. Suitable methods fortransforming Fusarium species are described by Malardier et al., 1989,Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using theprocedures described by Becker and Guarente, In Abelson, J. N. andSimon, M. I., editors, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Examples of suitable yeast host cells include strains of Saccharomyces,e.g. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such asP. pastoris or P. methlanolica, Hansenula, such as H. Polymorpha orYarrowia. Methods for transforming yeast cells with heterologous DNA andproducing heterologous polypeptides therefrom are disclosed by ClontechLaboratories, Inc, Palo Alto, Calif., USA (in the product protocol forthe Yeastmaker™ Yeast Tranformation System Kit), and by Reeves et al.,FEMS Microbiology Letters 99 (1992) 193-198, Manivasakam and Schiestl,Nucleic Acids Research, 1993, Vol. 21, No. 18, pp. 4414-4415 and Ganevaet al., FEMS Microbiology Letters 121 (1994) 159-164.

Examples of suitable insect host cells include a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells(High Five) (U.S. Pat. No. 5,077,214). Transformation of insect cellsand production of heterologous polypeptides therein may be performed asdescribed by Invitrogen.

Examples of suitable mammalian host cells include Chinese hamster ovary(CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines(COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells(e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 orATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well asplant cells in tissue culture. Additional suitable cell lines are knownin the art and available from public depositories such as the AmericanType Culture Collection, Rockville, Md. Also, the mammalian cell, suchas a CHO cell, may be modified to express sialyltransferase, e.g.1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, inorder to provide improved glycosylation of the IFNG polypeptide.

Methods for introducing exogenous DNA into mammalian host cells includecalcium phosphate-mediated transfection, electroporation, DEAE-dextranmediated transfection, liposome-mediated transfection, viral vectors andthe transfection method described by Life Technologies Ltd, Paisley, UKusing Lipofectamin 2000. These methods are well known in the art ande.g. described by Ausbel et al. (eds.), 1996, Current Protocols inMolecular Biology, John Wiley & Sons, New York, USA. The cultivation ofmammalian cells are conducted according to established methods, e.g. asdisclosed in (Animal Cell Biotechnology, Methods and Protocols, Editedby Nigel Jenkins, 1999, Human Press Inc, Totowa, N.J., USA and HarrisonM A and Rae I F, General Techniques of Cell Culture, CambridgeUniversity Press 1997).

In order to produce a glycosylated polypeptide a eukaryotic host cell,e.g. of the type mentioned above, is preferably used.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known n the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989). Specificmethods for purifying polypeptides exhibiting IFNG activity aredisclosed in EP 110044 and unexamined Japanese patent application No.186995/84.

The biological activity of the IFNG polypeptide can be assayed by anysuitable method known in the art. Such assays include antibodyneutralization of antiviral activity, induction of protein kinase,oligoadenylate 2,5-A synthetase or phosphodiesterase activities, asdescribed in EP 0 041 313 B1. Such assays also include immunomodulatoryassays (see, e.g., U.S. Pat. No. 4,753,795), growth inhibition assays,and measurement of binding to cells that express interferon receptors. Aspecific assay (entitled “primary Assay”) is described in the Materialsand Methods section herein.

Pharmaceutical Compositions and Uses Thereof

Furthermore, the present invention relates to improved methods oftreating, in particular, inflammatory diseases, e.g. interstitial lungdiseases, such as idiopathic pulmonary fibrosis, but also granulomatousdiseases; cancer, in particular ovarian cancer; infections such aspulmonary atypical mycobacterial infections; bone disorders (e.g. a bonemetabolism disorder so as malignant osteopetrosis); autoimmune diseasessuch as rheumatoid arthritis; as well as other diseases such asmultiresistent tuberculosis; cryptococcal meningitis; cystic fibrosisand liver fibrosis, in particular liver fibrosis secondary to hepatitisC; asthma; lymphoma; the key advantages being less frequent and/or lessintrusive administration of more efficient therapy, and optionally alower risk of immune reactions with the therapeutically activecompound(s).

The molecule of the invention is preferably administered in acomposition including a pharmaceutically acceptable carrier orexcipient. “Pharmaceutically acceptable” means a carrier or excipientthat does not cause any untoward effects in patients to whom it isadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]).

The molecules of the invention can be used “as is” and/or in a salt formthereof. Suitable salts include, but are not limited to, salts withalkali metals or alkaline earth metals, such as sodium, potassium,calcium and magnesium, as well as e.g. zinc salts. These salts orcomplexes may by present as a crystalline and/or amorphous structure.

The polypeptide of the invention is administered at a dose approximatelyparalleling that employed in therapy with known commercial preparationsof IFNG, such as Actimmune®, or as specified in EP 0 795 332. The exactdose to be administered depends on the circumstances. Normally, the doseshould be capable of preventing or lessening the severity or spread ofthe condition or indication being treated. It will be apparent to thoseof skill in the art that an effective amount of the IFNG polypeptide orcomposition of the invention depends, inter alia, upon the disease, thedose, the administration schedule, whether the polypeptide orcomposition is administered alone or in conjunction with othertherapeutic agents, the serum half-life/functional in vivo half-life ofthe compositions, and the general health of the patient.

The present invention also relates to an IFNG polypeptide according tothe present invention or a pharmaceutical composition according to thepresent invention for use as a medicament.

Furthermore, the invention also relates to the use of i) an IFNG variantaccording to the present invention, or ii) a pharmaceutical compositionof the invention, for the manufacture of a medicament, a pharmaceuticalcomposition or a kit-of-parts for the treatment of diseases selectedfrom the group consisting of inflammatory diseases, such as interstitiallung diseases, in particular idiopathic pulmonary fibrosis; cancer, inparticular ovarian cancer; infections, such as pulmonary atypicalmycobacterial infections; bone disorders (e.g. a bone metabolismdisorder so as malignant osteopetrosis); granulomatous diseases;autoimmune diseases such as rheumatoid arthritis; multiresistenttuberculosis; cryptococcal meningitis; cystic fibrosis and liverfibrosis, in particular liver fibrosis secondary to hepatitis C; asthmaand lymphoma. Most preferably the disease is an interstitial lungdisease, in particular idiopathic pulmonary fibrosis.

A glucocorticoid such as prednisolone may also be included. Thepreferred dosing is 1-4, more preferably 2-3, μg/kg patient weight ofthe polypeptide component per dose. The preferred dosing is 100-350,more preferably 100-150 μg glucocorticoid/kg patient weight per dose.

Also disclosed are improved means of delivering the molecules orpreparations, optionally additionally comprising glucocorticoids.

The invention also relates to a kit of parts suitable for the treatmentof interstitial lung diseases comprising a first pharmaceuticalcomposition comprising the active components i) or ii) mentioned aboveand a second pharmaceutical composition comprising at least oneglucocorticoid, each optionally together with a pharmaceuticallyacceptable carrier and/or excipient.

The variant of the invention can be formulated into pharmaceuticalcompositions by well-known methods. Suitable formulations are describedby Remington's Pharmaceutical Sciences by E. W. Martin and U.S. Pat. No.5,183,746.

The pharmaceutical composition may be formulated in a variety of forms,including liquid, gel, lyophilized, powder, compressed solid, or anyother suitable form. The preferred form will depend upon the particularindication being treated and will be apparent to one of skill in theart. However, the IFNG polypeptide of the invention is preferablyformulated as a liquid pharmaceutical composition.

The pharmaceutical composition may be administered orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner,e.g. using PowderJect or ProLease technology. The formulations can beadministered continuously by infusion, although bolus injection isacceptable, using techniques well known in the art, such as pumps orimplantation. In some instances the formulations may be directly appliedas a solution or spray. The preferred mode of administration will dependupon the particular indication being treated and will be apparent to oneof skill in the art, but usually subcutaneous administration ispreferred as this mode of administration can typically be conducted bythe patient himself.

The pharmaceutical composition of the invention may be administered inconjunction with other therapeutic agents. These agents may beincorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide of the invention, eitherconcurrently or in accordance with any other acceptable treatmentschedule. In addition, the polypeptide or pharmaceutical composition ofthe invention may be used as an adjunct to other therapies. Inparticular, combinations with glucocorticoids as described in EP 0 795332 are considered.

Parenterals

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM Suitable buffering agents for usewith the present invention include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween(®-80, etc.).

Additional miscellaneous excipietits include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

In a preferred embodiment of the invention said pharmaceuticalcomposition comprises the i) IFNG variant of the invention, ii) abuffering agent, in particular a salt of an organic acid, capable ofmaintaining the pH between 4.5-7.5, iii) a stabilizer, in particular anorganic sugar or sugar alcohol, iv) a non-ionic surfactant, and v)sterile water. Preferably, the buffering agent is capable of maintainingthe pH between 5.0-7.5, more preferably between 5.0-7.0, in particularbetween 5.0-6.5. More particularly, the buffering agent is selected fromthe group consisting of acetate, succinate and citrate, the stabilizeris mannitol or sorbitol, the non-ionic surfactant is Tween®-20 orTween®-80. Preferably, the pharmaceutical composition does not includeany preservatives.

In a highly preferred embodiment of the invention, the pharmaceuticalcomposition comprises an sulfoalkyl ether cyclodextrin derivative, suchas any of the derivatives described in U.S. Pat. No. 5,874,418, U.S.Pat. No. 5,376,645 and U.S. Pat. No. 5,134,127, the contents of whichare incorporated herein by reference. In one embodiment of the inventionthe sulfoalkyl ether cyclodextrin is a compound of the Formula (I):

wherein

-   -   n is 4, 5 or 6,    -   R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,        —O— or a —O—(C₂-C₆ alkylene)-SO₃— group, wherein at least one of        R₁ and R₂ is independently a —O—(C₂-C₆ alkylene)-SO₃— group, and        S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently,        a pharmaceutically acceptable cation.

In a further embodiment n is 5. In a still further embodiment n is 6.

In a further embodiment at least one of R₁ and R₂ is —O—(CH₂)_(m)—SO₃—,and m is 2, 3, 4, 5 or 6. In a further embodiment R₁ and R₂ isindependently selected from —OCH₂CH₂CH₂SO₃— or —OCH₂CH₂CH₂CH₂SO₃—.

In a further embodiment at least one of R₄, R₆, and R₈, isindependently, —O—(C₂-C₆ alkylene)-SO₃—; and R₅, R₇, and R₉ are all —O—.

In a further embodiment S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each,independently, a pharmaceutically acceptable cation selected from H⁺,alkali metals (e.g. Li⁺, Na⁺, K⁺), alkaline earth metals (e.g., Ca⁺²,Mg⁺²), ammonium ions and amine cations such as the cations of (C₁-C₆)alkylamines, piperidine, pyrazine, (C₁-C₆) alkanolamine and(C₄-C₈)cycloalkanolamine.

In a further embodiment S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areindependently selected from alkaline metal cation, alkaline earth metalcation, quaternary ammonium cation, tertiary ammonium cation, andsecondary ammonium cation.

In a further embodiment at least one of R₄, R₆, and R₈, isindependently, —O—(C₂-C₆ alkylene)-SO₃—; and R₅, R₇, and R₉ are all —O—.

The terms “alkylene” and “alkyl,” as used herein (e.g., in the—O—(C₂-C₆-alkylene)SO₃— group or in the alkylamines), include linear,cyclic, and branched, saturated and unsaturated (i.e., containing adouble bond) divalent alkylene groups and monovalent alkyl groups,respectively. The term “alkanol” in this text likewise includes bothlinear, cyclic and branched, saturated and unsaturated alkyl componentsof the alkanol groups, in which the hydroxyl groups may be situated atany position on the alkyl moiety. The term “cycloalkanol” includesunsubstituted or substituted (e.g., by methyl or ethyl) cyclic alcohols.

The presently preferred sulfoalkyl ether cyclodextrin derivative is asalt of beta cyclodextrin sulfobutyl ether (in particular the sodiumsalt thereof also termed SBE7-β-CD which is available as Captisol®)(Cydex, Overland Park, Kans. 66213, US).

Sustained Release Preparations

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thevariant, the matrices having a suitable form such as a film ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S-S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Oral Administration

For oral administration, the pharmaceutical composition may be in solidor liquid form, e.g. in the form of a capsule, tablet, suspension,emulsion or solution. The pharmaceutical composition is preferably madein the form of a dosage unit containing a given amount of the activeingredient. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, butcan be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules,tablets, suppositories, powders and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances, e.g. lubricatingagents such as magnesium stearate. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

The variants may be admixed with adjuvants such as lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, they may bedissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants such as preservatives, stabilizers, wettingagents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhereherein.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants such as wetting agents,sweeteners, flavoring agents and perfuming agents.

Topical Administration

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin(e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose.

Pulmonary Delivery

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the polypeptide dissolved in waterat a concentration of, e.g., about 0.01 to 25 mg of variant per mL ofsolution, preferably about 0.1 to 10 mg/mL. The formulation may alsoinclude a buffer and a simple sugar (e.g., for protein stabilization andregulation of osmotic pressure), and/or human serum albumin ranging inconcentration from 0.1 to 10 mg/ml. Examples of buffers that may be usedare sodium acetate, citrate and glycine. Preferably, the buffer willhave a composition and molarity suitable to adjust the solution to a pHin the range of 3 to 9. Generally, buffer molarities of from 1 mM to 50mM are suitable for this purpose. Examples of sugars which can beutilized are lactose, maltose, mannitol, sorbitol, trehalose, andxylose, usually in amounts ranging from 1% to 10% by weight of theformulation.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the protein caused by atomizationof the solution in forming the aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbitan fatty acid esters. Amounts will generallyrange between 0.001% and 4% by weight of the formulation. An especiallypreferred surfactant for purposes of this invention is polyoxyethylenesorbitan monooleate.

Specific formulations and methods of generating suitable dispersions ofliquid particles of the invention are described in WO 94/20069, U.S.Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S. Pat. No. 5,957,124,U.S. Pat. No. 5,934,272, U.S. Pat. No. 5,915,378, U.S. Pat. No.5,855,564, U.S. Pat. No. 5,826,570 and U.S. Pat. No. 5,522,385 which arehereby incorporated by reference.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid variant formulation and may alsocontain a stabilizer such as human serum albumin (ISA). Typically, morethan 0.5% (w/w) HSA is added. Additionally, one or more sugars or sugaralcohols may be added to the preparation if necessary. Examples includelactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, andxylose. The amount added to the formulation can range from about 0.01 to200% (w/w), preferably from approximately 1 to 50%, of the variantpresent. Such formulations are then lyophilized and milled to thedesired particle size.

The properly sized particles are then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C.

Formulations for powder inhalers will comprise a finely divided drypowder containing variant and may also include a bulking agent, such aslactose, sorbitol, sucrose, or mannitol in amounts which facilitatedispersal of the powder from the device, e.g., 50% to 90% by weight ofthe formulation. The particles of the powder shall have aerodynamicproperties in the lung corresponding to particles with a density ofabout 1 g/cm² having a median diameter less than micrometers, preferablybetween 0.5 and 5 micrometers, most preferably of between 1.5 and 3.5micrometers. An example of a powder inhaler suitable for use inaccordance with the teachings herein is the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

The powders for these devices may be generated and/or delivered bymethods disclosed in U.S. Pat. No. 5,997,848, U.S. Pat. No. 5,993,783,U.S. Pat. No. 5,985,248, U.S. Pat. No. 5,976574, U.S. Pat. No.5,922,354, U.S. Pat. No. 5,785,049 and U.S. Pat. No. 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.; the “standing cloud” device of InhaleTherapeutic Systems, Inc., San Carlos, Calif.; the AIR inhalermanufactured by Alkermes, Cambridge, Mass.; and the AERx pulmonary drugdelivery system manufactured by Aradigm Corporation, Hayward, Calif.

The invention provides compositions and methods for treating bacterialand viral infections, cancers or tumors, interstitial pulmonary diseasessuch as idiopathic pulmonary fibrosis, granulomatous diseases, bonedisorders (e.g. a bone metabolism disorder so as malignantosteopetrosis) and autoimmune diseases such rheumatoid arthritis.

In a further aspect the invention relates to a method of treating amammal having circulating antibodies against huIFNG, which methodcomprises administering a compound which has the bioactivity of IFNG andwhich does not react with said antibodies. The compound is preferably avariant as described herein and the mammal is preferably a human being.The mammals to be treated may suffer from any of the diseases listedabove for which IFNG is a useful treatment. Furthermore, the inventionrelates to a method of making a pharmaceutical product for use intreatment of mammals having circulating antibodies against huIFNG,wherein a compound which has the bioactivity of IFNG and which does notreact with such is formulated into an injectable or otherwise suitableformulation. The term “circulating antibodies” is intended to indicateautoantibodies formed in a mammal in response to having been treatedwith any of the commercially available IFNG preparations.

Also contemplated is use of a nucleotide sequence encoding an IFNGpolypeptide of the invention in gene therapy applications. Inparticular, it may be of interest to use a nucleotide sequence encodingan IFNG polypeptide described in the section above entitled“Glycosylated Polypeptides of the Invention modified to incorporateadditional glycosylation sites”. The glycosylation of the polypeptide isthus achieved during the course of the gene therapy, i.e. afterexpression of the nucleotide sequence in the human body.

Gene therapy applications contemplated include treatment of thosediseases in which the polypeptide is expected to provide an effectivetherapy.

Local delivery of IFNG using gene therapy may provide the therapeuticagent to the target area while avoiding potential toxicity problemsassociated with non-specific administration.

Both in vitro and in vivo gene therapy methodologies are contemplated.

Several methods for transferring potentially therapeutic genes todefined cell populations are known. For further reference see, e.g.,Mulligan, “The Basic Science Of Gene Therapy”, Science, 260, pp. 926-31(1993). These methods include:

Direct gene transfer, e.g., as disclosed by Wolff et al., “Direct Genetransfer Into Mouse Muscle In vivo”, Science 247, pp. 1465-68 (1990);

Liposome-mediated DNA transfer, e.g., as disclosed by Caplen et al.,“Uposome-mediated CFFR Gene Transfer to the Nasal Epithelium Of PatientsWith Cystic Fibrosis” Nature Med., 3, pp. 39-46 (1995); Crystal, “TheGene As A Drug”, Nature Med., 1, pp. 15-17 (1995); Gao and Huang, “ANovel Cationic Liposome Reagent For Efficient Transfection of MammalianCells”, Biochem. Biophys Res. Comm., 179, pp. 280-85 (1991);

Retrovirus-mediated DNA transfer, e.g., as disclosed by Kay et al., “Invivo Gene Therapy of Hemophilia B: Sustained Partial Correction InFactor IX-Deficient Dogs”, Science, 262, pp. 117-19 (1993); Anderson,“Human Gene Therapy”, Science, 256, pp.808-13(1992);

DNA Virus-mediated DNA transfer. Such DNA viruses include adenoviruses(preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferablyherpes simplex virus based vectors), and parvoviruses (preferably“defective” or non-autonomous parvovirus based vectors, more preferablyadeno-associated virus based vectors, most preferably AAV-2 basedvectors). See, e.g., Ali et al., “The Use Of DNA Viruses as Vectors forGene Therapy”, Gene Therapy, 1, pp. 367-84 (1994); U.S. Pat. No.4,797,368, and U.S. Pat. No. 5,139,941.

The invention is further described in the following examples. Theexamples should not, in any manner, be understood as limiting thegenerality of the present specification and claims.

Materials and Methods

Materials

-   CHO-K1 cells (available from American Type Culture Collection (ATCC    #CCL-61)).-   HeLa cells (available from American Type Culture Collection (ATCC    #CCL-2)).-   ISRE-Luc was obtained from Stratagene, La Jolla USA.-   pCDNA 3.1/hygro was obtained from Invitrogen, Carlsbad USA.-   Restriction enzymes and polymerases were obtained from New England    Biolabs Inc., Beverly, USA.-   DMEM medium: Dulbecco's Modified Eagle Media (DMEM), 10% fetal    bovine serum and-   Hygromycin B were obtained from Life Technologies A/S, Copenhagen,    Denmark.-   LucLite substrate was obtained from Packard Bioscience, Groningen,    The Netherlands.-   TopCount luminometer was obtained from Packard Bioscience,    Groningen, The Netherlands.-   Biotinylated polyclonal anti-human IFNG antibody, BAF285, was    obtained available from R&D Systems Inc., Minneapolis, USA.-   Horse Radish Peroxidase-conjugated streptavidin, P0397, was obtained    from DAKO, Copenhagen, Denmark.-   TMB blotting reagent was obtained from KEM-EN-TEC, Copenhagen,    Denmark.    Methods    Interferon Assay Outline

It has previously been published that IFNG interacts with and activatesIFNG receptors on HeLa cells. Consequently, transcription is activatedat promoters containing an Interferon Stimulated Response Element(ISRE). It is thus possible to screen for agonists of interferonreceptors by use of an ISRE coupled luciferase reporter gene (ISRE-luc)placed in HeLa cells.

Primary Assay

HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygro and foci(cell clones) are created by selection in DMEM media containingHygromycin B. Cell clones are screened for luciferase activity in thepresence or absence of IFNG. Those clones showing the highest ratio ofstimulated to unstimulated luciferase activity are used in furtherassays.

To screen polypeptides, 15,000 cells/well are seeded in 96 well cultureplates and incubated overnight in DMEM media. The next day thepolypeptides as well as a known standard are added to the cells invarious concentrations. The plates are incubated for 6 hours at 37° C.in a 5% CO₂ air atmosphere LucLite substrate (Packard Bioscience,Groningen, The Netherlands) is subsequently added to each well. Platesare sealed and luminescence measured on a TopCount luminometer (Packard)in SPC (single photon counting) mode.

Each individual plate contains wells incubated with IFNG as a stimulatedcontrol and other wells containing normal media as an unstimulatedcontrol. The ratio between stimulated and unstimulated luciferaseactivity serves as an internal standard for both IFNG activity andexperiment-to-experiment variation.

Determination of Increased Degree of Glycosylation

To determine the various degrees of glycosylation of IFNG monomers, aSDS-PAGE gel is run under standard conditions and transferred to anitrocellulose membrane. Western blotting is done according to standardprocedures using a biotinylated polyclonal anti-human IFNG antibody(BAF285 from R & D Systems) as primary antibody and Horse RadishPeroxidase-conjugated streptavidin (P0397from DAKO) as secondaryantibody followed by staining with TNB blotting reagent (KEM-EN-TEC,Copenhagen, Denmark). The distribution of IFNG monomers having varyingdegrees of glycosylation is made by visual inspection of the stainedmembrane.

Determination of AUC_(sc)

The AUC_(sc) is determined by one 200 μl bolus subcutaneousadministration of equal amount (on an activity basis) of the IFNGpolypeptide of the invention in rats.

For these experiments, female Sprag-Dawley rats, weighing between220-260 grams, are used. The IFNG polypeptide is formulated in sodiumsuccinate (720 mg/l), mannitol 40 g/l), polysorbat 20 (100 mg/l) at pH6.0.

Before subcutaneous administration, one blood sample is drawn in thetail-vein to ensure that no background IFNG activity can be detected.After administration, blood samples are withdrawn from the tail veinafter 10 min, 20 min, 40 min, 60 min, 120 min, 240 min, 480 min, 720min, 1440 min, 1620 min, 1920 min and 2880 min (sometimes also 3600min). Serum is prepared by letting the blood sample coagulate for 20 minat room temperature followed by centrifugation at 5000 g, 20 min at roomtemperature. The serum is then isolated and stored at −80° C. untildetermination of IFNG activity using the “Primary Assay” describedabove.

The amount of units in serum (U/ml) against time (min) is then plottedand the AUC_(sc) is calculated using GraphPad Prism 3.01.

Similar experiments are performed on glycosylated huIFNG, glycosylated[S99T]huIFNG and/or Actimmune® in order to assess the increase inAUC_(sc) of the IFNG polypeptide of the invention as compared toglycosylated huIFNG, glycosylated [S99T]huIFNG and/or Actimmune®.

Functional in Vivo Half-Life

The functional in vivo half-life is determined by one 200 μl bolusintravenous administration of equal amount (on an activity basis) of theIFNG polypeptide of the invention in rats.

For these experiments, female Sprag-Dawley rats, weighing between220-260 grams, are used. The IFNG polypeptide is formulated in sodiumsuccinate (720 mg/l), mannitol 40 g/l), polysorbat 20 (100 mg/l) at pH6.0.

Before intravenous administration, one blood sample is drawn in thetail-vein to ensure that no background IFNG activity can be detected.After administration in one tail vein, blood samples are withdrawn fromthe other tail vein after 5 min, 10 min, 20 min, 40 min, 60 min, 120min, 240 min, 480 min, 720 min, 1440 min, 1620 min, 1920 min and 2880min. Serum is prepared by letting the blood sample coagulate for 20 minat room temperature followed by centrifugation at 5000 g, 20 min at roomtemperature. The serum is then isolated and stored at −80° C. untildetermination of IFNG activity using the “Primary Assay” describedabove.

The amount of units in serum (U/ml) against time (min) is then plottedand the functional in vivo half-life is calculated using WinNonLin Pro3.3.

Similar experiments are performed on glycosylated huIFNG, glycosylated[S99T]huIFNG and/or Actimmune® in order to assess the increase infunctional in vivo half-life of the IFNG polypeptide of the invention ascompared to glycosylated huIFNG, glycosylated [S99T]huIFNG and/orActimmune®.

Identification of Surface Exposed Amino Acid Residues

Structures

Experimental 3D structures of huIFNG determined by X-ray crystallographyhave been reported by: Ealick et al. Science 252:698-702 (1991)reporting on the C-alpha trace of an IFNG homodimer. Walter et. al.Nature 376:230-235 (1995) reporting on the structure of an IFNGhomodimer in complex with two molecules of a soluble form of the IFNGreceptor. The coordinates of this structure have never been madepublicly available. Thiel et. al. Structure 8:927-936 (2000) reportingon the structure of an IFNG homodimer in complex with two molecules of asoluble form of the IFNG receptor having a third molecule of thereceptor in the structure not making interactions with the IFNGhomodimer.

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (Copyright (c) 1983 Yale University) wasused to compute the accessible surface area (ASA) of the individualatoms in the structure. This method typically uses a probe-size of 1.4 Åand defines the Accessible Surface Area (ASA) as the area formed by thecentre of the probe. Prior to this calculation all water molecules,hydrogen atoms and other atoms not directly related to the protein areremoved from the coordinate set.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain with a valuerepresenting the ASA of the side chain atoms of that residue type in anextended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991)J. Mol. Biol.: 220, 507-530. For this example the CA atom is regarded asa part of the side chain of Glycine residues but not for the remainingresidues. The following table are used as standard 100% ASA for the sidechain: Ala 69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06 Å² Cys 96.69Å² Gln 140.58 Å² Glu 134.61 Å² Gly 32.28 Å² His 147.00 Å² Ile 137.91 Å²Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å² Pro 119.65 Å²Ser 78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions.

Determining Distances Between Atoms:

The distance between atoms was determined using molecular graphicssoftware e.g. InsightII v. 98.0, MSI INC.

Determination of Receptor Binding Site:

The receptor-binding site is defined as comprising of all residueshaving their accessible surface area changed upon receptor binding. Thisis determined by at least two ASA calculations; one on the isolatedligand(s) in the ligand(s)/receptor(s) complex and one on the completeligand(s)/receptor(s) complex.

EXAMPLES Example 1 Determination of Surface-Exposed Amino Acids

The X-ray structure used was of an IFNG homo-dimer in complex with twomolecules of a soluble form of the IFNG receptor having a third moleculeof the IFNG receptor in the structure not making interactions with theIFNG homodimer reported by Thiel et. al. Structure 8:927-936 (2000). Thestructure consists of the IFNG homodimer wherein the two molecules arelabeled A and B. For construction purposes there is an additionalmethionine placed before the IFNG sequence labeled MO and the sequenceis C-terminally truancuted with ten residues (Q133 being the lastresidue in the constructed molecules). The MO is removed from thestructure in all the calculations of this example. The structure of thetwo IFNG monomers has very weak electron density after residue 120 andresidues were only modeled until residue T126. Therefore, residuesS121-T126 were removed from the structure prior to the calculations inthis example. The two receptor fragments labeled C and D make directinteractions with the IFNG homodimer and a third receptor moleculelabeled E makes no contact with the IFNG homodimer and are not includedin these calculations.

Surface Exposure:

Performing fractional ASA calculations on the homodimer of molecules Aand B excluding MO and S121-T126 in both molecules resulted in thefollowing residues having more than 25% of their side chain exposed tothe surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10,K12, K13, Y14, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31,K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63, Q64, S65,Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87,D90, E93, K94, N97, S99, T101, D102, L103, N104, H11, Q115, A118 andE119.

The following residues had more than 50% of their side chain exposed tothe surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10,K13, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31, K34, K37,E38, E39, K55, K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86,K87, K94, N97, S99, T101, D102, L103, N104, Q115, A118, E119.

Performing fractional ASA calculations on the homodimer of molecules Aand B excluding MO and S121-T126 in both molecules and including thereceptor molecules C and D resulted in the following residues had morethan 25% of their side chain exposed to the surface in at least one ofthe monomers: Q1, D2, P3, K6, E9, N10, K13, Y14, N16, G18, H19, D21,N25, G26, G31, K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62,D63, Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84,N85, K86, K87, D90, E93, K94, N97, S99, T101, D102, L103, N104, E119.

The following residues had more than 50% of their side chain exposed tothe surface in at least one of the monomers: P3, K6, N10, K13, N16, D21,N25, G26, G31, K34, K37, E38, E39, K55, K58, N59, D62, Q64, S65, K68,E71, E75, N83, S84, K86, K87, D90, E93, K94, T101, D102, L103 and N104.

All of the above positions are targets for modification in accordancewith the present invention.

Comparing the two lists, results in K12, S20, A23, D24, T27, H111, Q115and A118 being removed from the more than 25% side chain ASA list uponreceptor binding, and Q1, D2, E9, G18, H19, S20, A23, D24, T27, Q 15,A118 and E119 being removed from the more t 50% side chain ASA list uponreceptor binding.

Residues not determined in the structure are treated as fully surfaceexposed, i.e. residues S121, P122, A123, A124, K125, T126, G127, K128,R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140,A141, S142, Q143. These residues also constitute separate targets forintroduction of attachment groups in accordance with the presentinvention (or may be viewed as belonging to the group of surface exposedamino acid residues, e.g. having more than 25% or more than 50% exposedside chains).

Example 2 Determination of Receptor Binding Site

Performing ASA calculations as described above results in the followingresidues of the IFNG molecule having reduced ASA in at least one of themonomers in the complex as compared to the calculation on the isolateddimer: Q1, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25,G26, T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119.

Example 3 Design of an Expression Cassette for Expression of IFNG withCodon Usage Optimised for CHO Cells

The DNA sequence, GenBank accession number X13274, encompassing a fulllength cDNA encoding mature huIFNG with its native signal peptide, wasmodified in order to facilitate high expression in CHO cells. Codons ofthe huIFNG nucleotide sequence were modified by making a bias in thecodon usage towards the codons frequently used in homo sapiens.Subsequently, certain nucleotides in the sequence were substituted withothers in order to introduce recognition sites for DNA restrictionendonucleases. Primers were designed such that the gene could besynthesised.

The primers were assembled to the synthetic gene by one step PCR usingPlatinum Pfx-polymerase kit (Life Technologies) and standard three-stepPCR cycling parameters. The assembled gene was amplified by PCR usingthe same conditions and has the sequence shown in SEQ ID NO:4. Thesynthesised gene was cloned into pcDNA3.1/hygro (InVitrogen) between theBamHI at the 5′ end and the XbaI at the 3′ end, resulting in pIGY-22.

Example 4 Site Directed Mutagenesis

Generation of N-glycosylation Variants

To introduce mutations/optimise N-glycosylation sites in IFNG,oligonucleotides were designed in such a way that PCR generated changescould be introduced in the expression plasmid (pIGY-22) by classicaltwo-step PCR followed by subcloning the PCR fragment using BamHI andXbaI.

Two vector primers were designed to be used with specific mutationprimers: ADJ013: 5′-GATGGCTGGCAACTAGAAG-3′ (antisense downstream vectorprimer) ADJ014: 5′-TGTACGGTGGGAGGTCTAT-3′ (sense upstream vector primer)

To optimise the native N-glycosylation site at position 97 and in orderto introduce an additional N-glycosylation site at position 38, thefollowing primers were designed: S99T: ADJ093 5′-GTTCAGGTCTGTCACGGTGTAATTGGTCAGCTT-3′ ADJ094 5′-AAGCTGACCAATTACACCGTGACAGACCTGAAC-3 E38N + S40T: ADJ091 5′-CATGATCTTCCGATCGGTCTCGTTCTTCCAATT-3′ ADJ092 5′-AATTGGAAGAACGAGACCGATCGGAAGATCATG-3′

The S99T variant was generated by classical two-step PCR as describedabove, using ADJ013 and ADJ014 as vector primers, ADJ093 and ADJ094 asmutation primers, and pIGY-22 as template. The 447 bp PCR fragment wassubcloned into pcDNA3.1/Hygro (InVitrogen) using BamHI and XbaI, leadingto plasmid pIGY48.

The E38N+S40T+S99T variant was generated by classical two-step PCR asdescribed above, using ADJ013 and ADJ014 as vector primers, ADJ091 andADJ092 as mutation primers, and pIGY-48 as template. The 447 bp PCRfragment was subcloned into pcDNA3.l/Hygro (InVitrogen) using BamHI andXbaI, leading to plasmid pIGY-54.

Generation of the C-Terminally Modified IFNG Variants

C-terminally modified IFNG variants were generated by one-step PCR usingpIGY-54 as template (i.e. including the E38N+S40T+S99T mutations). An‘upstream’ oligonucleotide containing the start codon (preceded by aBamHI site for cloning and the sequence GCCGCCACC in order to optimisemRNA translation) and a ‘downstream’ oligonucleotide containing thedesired mutation(s) and a XbaI site were used as primers.

In order to optimise protein production, apcDNA3.1(+)/Hygro(InVitrogen)—derivative plasmid containing an intronfrom pCI-Neo (Stratagene) was used as expression vector. This vector,termed PF033, was constructed by PCR amplification of the intron frompCI-Neo using 5′-CCGTCAGATCCTAGGCTAGCTTATTGCGGTAGTTTATCAC-3′ and5′-GAGCTCGGTACCAAGCTTTTAAGAGCTGTAAT-3′ as primers, followed bysubcloning of the PCR product into pcDNA3.1(+)/Hygro using NheI andHindIII. The mutated IFNG PCR products were subcloned into PF033 usingBamHI and XbaI.

Using this approach the following variants were prepared:

-   E38N+S40T+S99T+R137P (reference)-   E38N+S40T+S99T+R139P (reference)-   E38N+S40T+S99T+R140P (reference)-   E38N+S40T+S99T+S142P (reference)-   E38N+S40T+S99T+R137P+R139P+Q143P (reference)-   E38N+S40T+S99T+R137P+R139P+S142P-   E38N+S40T+S99T+R137P+S142P-   E38N+S40T+S99T+S132P+R137P+R140P-   E38N+S40T+S99T+S132P+R140P-   E38N+S40T+S99T+R140P (reference)-   E38N+S40T+S99T+R137P+R140P

Example 5 Expression of IFNG Polypeptides in Mammalian Cells

For transient expression of the IFNG polypeptide, cells were grown to95% confluency in serum-containing media (Dulbecco's MEM/Nut.-mix F-12(Ham) L-glutamine, 15 mM Hepes, pyridoxine-HCl (Life Technologies Cat #31330-038)) containing 1:10 FBS (BioWhittaker Cat # 02-701F) and 1:100penicillin and streptomycin (BioWhittaker Cat # 17-602E). IFNG-encodingplasmids were transfected into the cells using Lipofectamine 2000 WifeTechnologies) according to the manufacturer's specifications. 24 hrsafter transfection, culture media were collected and assayed for IFNGactivity. Furthermore, in order to quantify the relative number ofglycosylation sites utilized, Western blotting is performed usingharvested culture medium.

Stable clones expressing the IFNG polypeptide are generated bytransfection of CHO K1 cells with IFNG-encoding plasmids followed byincubation of the cells in media containing 0.36 mg/ml hygromycin.Stably transfected cells are isolated and sub-cloned by limiteddilution. Clones producing high levels of IFNG are identified by ELISA.

Example 6 Large-Scale Production

Stable cell lines expressing the IFNG polypeptide are grown inDulbecco's MEM/Nut.-mix F-12 (Ham) L-glutamine, 15 mM Hepes,pyridoxine-HCl (Life Technologies Cat # 31330-038), 1:10 FBS(BioWhittaker Cat # 02-701F), 1:100 penicillin and streptomycin(BioWhittaker Cat # 17-602E) in 1700 cm2 roller bottles (Corning, #431200) until confluence. The media is then changed to 300 ml UltraCHOwith L-glutamine (BioWhittaker Cat # 12-724Q) with the addition of 1:500EX-CYTE VLE (Serological Proteins Inc. # 81-129) and 1:100 penicillinand streptomycin (BioWhittaker Cat # 17-602E). After 48 hours of growth,the media is replaced with fresh UltraCHO with the same additives. Afteranother 48 hours of growth, the media is replaced with Dulbecco'sMEM/Nut.-mix F-12 (Ham) L-glutamine, pyridoxine-HCl (Life TechnologiesCat # 21041-025) with the addition of 1:500 ITS-A (Gibco/BRL#51300-044), 1:500 EX-CYTE VIE (Serological Proteins Inc. # 81-129) and1:100 penicillin and streptomycin (BioWhittaker Cat # 17-602E).Subsequently, every 24 h, culture media are harvested and replaced with300 ml of the same serum-free media. The collected media are filteredthrough 0.22 μm filters to remove cells.

Example 7 Purification

The filtrate was microfiltrated (0.22 μm) before ultrafiltration toapproximately 1/20 volume using a Millipore Liz system. On the samesystem the concentrate was diafiltrated using 10 mM Tris, pH 7.6.Ammonium sulphate was added to a concentration of 2.1 M and afterstirring the precipitate was removed by centrifugation at 8000 rpm for25 minutes in a Sorvall centrifuge using a GS3 rotor.

The supernatant was applied onto an Ether 650M (Toyoperl, Tosohaas)column previously equilibrated in 10 mM Tris, 2.1 M ammonium sulphate,pH 7.6. The flow-through fraction containing the IFNG variant was loadeddirectly onto a butyl-sepharose FF (Amersham Biosciences) columnpre-equilibrated with, 2.1 M ammonium sulphate, pH 7.6. The column waswashed with 2 column volumes of 10 mM Tris, 2.1 M ammonium sulphate, pH7.6 after application and the bound IFNG variant was then eluted in alinear gradient over 20 column volumes to 100% 10 mM Tris, pH 7.6.Fractions enriched in the IFNG variant were pooled and buffer exchangedby diafiltration into 10 mM Tris, pH 8.8, using a Vivaflow200 system(VivaScience) with a molecular weight cut-off of 10,000 Da.

The IFNG variant was then applied onto a Q-sepharose FF (AmershamBiosciences) column previously equilibrated in 10 mM Tris, pH 8.8. Afterapplication the column was washed with 3 column volumes of 10 mM Tris,pH 8.8 before eluting the bound IFNG variant in a gradient from 0-30% 10mM Tris-HCl, 1 M NaCl, pH 8.8, over 25 column volumes. Fractionscontaining the IFNG variant were pooled and buffer exchanged into 5 mMsodium succinate, 4% mannitol, pH 6.0, using a VivaSpin2o column(VivaScience) and Tween 20 was subsequently added to a concentration of0.01%. The IFNG variant was sterile filtered and stored at −80° C.

Example 8 Analysis of C-Terminal Truncation

The above-mentioned variants were constructed to study the C-terminaltruncation in more detail. The variants were purified from serum-freemedia as described in Example 5, except that stabile clones from pooledclones were used instead of selected high-expressing single clones. Ingeneral, 2500 to 5000 ml media was used to purify the individualvariants.

The sterile-filtered media (0.22 μm) were concentrated to approx. 1/15volume and subsequently diafiltered (to a conductivity <2 mS/cm) using 5mM sodium phosphate, pH 6.2, on a PALL FILTRON system. Theconcentrated/diafiltered media was filtered (0.22 μm) to clear thesample from any precipitated material prior to further purification. ThepH in the filtrate was adjusted to 6.2 before application onto a 2 mlCM-sepharose Fast Flow (Pharmacia) previously equilibrated in 10 mMsodium phosphate, pH 6.2. The column was washed with 10-15 columnvolumes 10 mM sodium phosphate, pH 6.2, before step-eluting boundvariants with 2-3 column volumes 100 mM sodium phosphate, 500 mM NaCl,pH 7.0.

The step-eluted variants were filtered (0.45 μm) before beingimmunoprecipitated with an IFNG antibody affinity column. The antibodyaffinity column was prepared according to the manufacture's instructionsby coupling 10 mg monoclonal mouse anti-human IFNG antibody (catalog no.MD-2, U-CyTech, Holland) onto approx. 1.3 ml activated CNBr-sepharose(Pharmacia). The filtered sample from the CM-sepharose column wasapplied onto the antibody affinity column previously equilibrated withphosphate-buffered saline. The column was then washed with 5 columnvolumes phosphate buffered saline and the variant was subsequentlyeluted into a vial already containing 0.15 ml 500 mM sodium phosphate,pH 7.2, using 2 column volumes 100 mM glycine, pH 3.5.

Analysis of C-terminal truncation was done by MALDI-TOF massspectrometry following enzymatic deglycosylation using the followingprocedure:

N-linked carbohydrate moieties attached to Asn-residues were removed bytreatment of 30 μl of the affinity-purified variant with 1 mU PNGase F(Roche) at 37° C. for 16 h. A 1 μl sample aliquot was mixed with 1 μlmatrix solution (saturated α-cyano-4-hydroxy cinnamic acid in 50%acetonitril, 0.1% TFA). Half of the mixture was spotted onto aThin-Layer of α-cyano-4-hydroxy cinnamic acid on the target plate andair-dried. (The Thin-Layer coating was preformed on the target plate bycrystallisation of α-cyano-4-hydroxy cinnamic acid from the Thin-Layermatrix solution (saturated α-cyano-4-hydroxy cinnamic acid in 100%acetone)). The sample spot was washed twice with 1 μl HPLC grade waterand air-dried. The sample spot was added 0.2 μl matrix solution andair-dried. Following introduction of the target plate into the massspectrometer, spectra were recorded at threshold laser power.

MALDI-TOF mass spectrometry was carried out in an Applied BiosystemsVoyager-DE Pro mass spectrometer in linear mode using positiveionisation. C-terminal truncation was evaluated by comparing recordedspectra with the expected mass of the purified variant.

Using the above approach, the following IFNG variants were analysed:

-   E38N+S40T+S99T (reference)—see FIG. 1-   E38N+S40T+S99T+R137P (reference)—see FIG. 2-   E38N+S40T+S99T+R139P (reference)—see FIG. 3-   E38N+S40T+S99T+S142P (reference)—see FIG. 4-   E38N+S40T+S99T+Q143P (reference)—see FIG. 5-   E38N+S40T+S99T+R137P+R139P (reference)—see FIG. 6-   E38N+S40T+S99T+R137P+R139P+Q143P (reference)—see FIG. 7-   E38N+S40T+S99T+R137P+R139P+S142P—see FIG. 8-   E38N+S40T+S99T+R137P+S142P—see FIG. 9-   E38N+S40T+S99T+S132P+R137P+R140P—see FIG. 10-   E38N+S40T+S99T+S132P+R140P—see FIG. 11-   E38N+S40T+S99T+R140P (reference)—see FIG. 12-   E38N+S40T+S99T+R137P+R140P—see FIG. 13

In FIG. 1 a significant C-terminal processing is clearly seen for thevariant [E38N+S40T+S99T]huIFNG.

In FIGS. 2 and 3 it can be seen that the presence of either of theadditional substitutions R137P or R139P leads to lesser, but stillpronounced, C-terminal processing than seen for [E38N+S40T+S99T]huIFNG.

In FIGS. 4 and 5 it can be seen that the presence of either of theadditional substitutions S142P or Q143P leads to unchanged, or even morepronounced, C-terminal processing than seen for [E38N+S40T+S99T]huIFNG.

In FIG. 6 it can be seen that the presence of both of the additionalsubstitutions R137P and R139P leads to lesser and more homogeneousC-terminal processing than seen for [E38N+S40T+S99T]huIFNG and for[E38N+S40T+S99T+R139P]huIFNG. The only C-terminally processed form thatcan be recognised for [E38N+S40T+S99T+R137P+R139P]huIFNG is the proteinhaving Ala141 as C-terminal amino acid residue.

FIG. 7 shows that further inclusion of the substitution Q143P in[E38N+S40T+S99T+R137P+R139P]huIFNG does not alter the C-terminalprocessing notably. The only C-terminally processed form that can berecognised for E38N+S40T+S99T+R137P+R139P+Q143P]huIFNG is still theprotein having Ala141 as C-terminal amino acid residue.

FIG. 8, however, shows that further inclusion of the substitution S142Pin [E38N+S40T+S99T+R137P+R139P]huIFNG alters the C-terminal processingnotably as the only protein present in significant amount is thefull-length protein having Q143 as the C-terminal amino acid residue.

FIG. 9 clearly shows that inclusion of the substitution S142P in[E38N+S40T+S99T+137P]huIFNG leads to a significantly smaller degree ofC-terminal processing and that the full-length protein having Q143 asthe C-terminal amino acid residue is the major species present (compareto FIG. 2).

FIG. 10 evidently shows that the variant[E38N+S40T+S99T+S132P+R137P+R140P] huIFNG is hardly C-terminallyprocessed. Only trace amounts of other species than the full-lengthprotein can be identified.

In a similar way, FIG. 11 shows that the full-length form of the variant[E38N+S40T+S99T+S132P+S140P]huIFNG is the major species.

In FIG. 12 a significant C-terminal processing is clearly seen for thevariant [E38N+S40T+S99T+R140P]huIFNG.

FIG. 13, however, shows that further inclusion of the substitution R140Pin [E38N+S40T+S99T+R137P]huIFNG alters the C-terminal processing notablyas the only protein present in significant amount is the full-lengthprotein having Q143 as the C-terminal amino acid residue.

1. A full-length variant of the interferon gamma (IFNG) polypeptide ofSEQ ID NO:1, said variant exhibiting IFNG activity and consisting of upto 10 residue modifications from residues 1 through 131 of SEQ ID NO:1,and (a) at least one amino acid substitution in a position selected fromthe group consisting of S132 and S142; and (b) at least one amino acidsubstitution in a position selected from the group consisting of R137,R139 and R140.
 2. The full-length variant according to claim 1, whereinsaid amino acid substitution is selected from the group consisting ofS132P, S142P and S132P+S142P.
 3. The full-length variant according toclaim 2, wherein said amino acid substitution is S132P.
 4. Thefull-length variant according to claim 2, wherein said amino acidsubstitution is S142P.
 5. The full-length variant of claim 2, wherein atleast one non-positively charged amino acid residue is introduced bysubstitution in a position selected from the group consisting of R137,R139 and R140.
 6. The full-length variant according to claim 5, whereinsaid non-positively charged amino acid residue is a proline residue. 7.The full-length variant of claim 5, wherein said variant comprises thefollowing substitutions: R137P+R139P+S142P.
 8. The full-length variantof claim 5, wherein said variant comprises the following substitutions:R137P+S142P.
 9. The full-length variant of claim 5, wherein said variantcomprises the following substitutions: S132P+R137P+R140P.
 10. Thefull-length variant of claim 5, wherein said variant comprises thefollowing substitutions: S132P+R140P.
 11. A full-length variant of theinterferon gamma (IFNG) polypeptide of SEQ ID NO:1. said variantexhibiting IFNG activity and consisting of up to 10 residuemodifications from residues 1 through 131 of SEQ ID NO:1, an amino acidsubstitution in position R137 and an amino acid substitution in positionR140.
 12. The full-length variant according to claim 11, wherein saidvariant comprises the substitutions R137X+R140P, wherein X is any aminoacid residue, except arginine and lysine.
 13. The full-length variantaccording to claim 11, wherein said variant comprises the substitutionsR137P+R140X, wherein X is any amino acid residue, except arginine. 14.The full-length variant of claim 11, wherein said variant comprises thesubstitutions R137P+R140P.
 15. The full-length variant of claim 11,wherein said variant comprises at least one further modification in theC-terminal part from amino acid residue S132 to amino acid residue Q143.16. The full-length variant according to claim 15, wherein said furthermodification comprises introduction of at least one cysteine residue.17. The full-length variant according to claim 16, wherein said cysteineresidue is covalently attached to a polymer molecule.
 18. Thefull-length variant according to claim 17, where said polymer moleculeis a linear or branched polyethylene glycol.
 19. (canceled)
 20. Thefull-length variant according to claim 11, wherein said modificationsare substitutions.
 21. The full-length variant according to claim 20,wherein said variant comprises the substitution S99T.
 22. Thefull-length variant of claim 1, wherein said up to 10 residuemodifications from residues 1 through 131 comprises at least oneintroduced and/or at least one removed amino acid residue comprising anattachment group for a non-polypeptide moiety.
 23. The full-lengthvariant according to claim 22, wherein said up to 10 residuemodifications comprises at least one introduced glycosylation site. 24.The full-length variant according to claim 23, wherein saidglycosylation site is an N-glycosylation site.
 25. The full-lengthvariant according to claim 24, wherein said N-glycosylation site isintroduced in a position comprising an amino acid residue having atleast 25% of its side chain exposed to the surface (as defined inExample 1 herein).
 26. The full-length variant according to claim 25,wherein said N-glycosylation site is introduced in a position comprisingan amino acid residue having at least 50% of its said chain exposed tothe surface (as defined in Example 1 herein).
 27. The full-lengthvariant of claim 24, wherein said N-glycosylation site is introduced bysubstitution.
 28. The full-length variant according to claim 1, whereinsaid up to 10 residue modifications is a substitution is selected fromthe group consisting of G18S, G18T, E38N, E38N+S40T, K61S, K61T,S65N+Q67S, S65N+Q67T, N85S, N85T, K94N, Q106S and Q106T.
 29. Thefull-length variant according to claim 28, wherein said substitution isselected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T,N85T, K94N and Q106T.
 30. The full-length variant according to claim 29,wherein said substitution is selected from the group consisting of G18T,E38N+S40T, K61T, S65N+Q67T and N85T.
 31. The full-length variantaccording to claim 30, wherein said substitution is E38N+S40T.
 32. Thefull-length variant according to claim 22, wherein said up to 10 residuemodifications comprises an introduced cysteine residue.
 33. Thefull-length variant according to claim 32, wherein said cysteine residueis introduced in a position comprising an amino acid residue having atleast 25% or its side chain exposed to the surface (as defined inExample 1 herein).
 34. The full-length variant according to claim 33,wherein said cysteine residue is introduced in a position comprising anamino acid residue having at least 50% or its side chain exposed to thesurface (as defined in Example 1 herein).
 35. The full-length variantaccording to any of claim 32, wherein said cysteine residue isintroduced by substitution.
 36. The full-length variant according toclaim 32, wherein said up to 10 residue modifications is a substitutionis selected from the group consisting of N10C, N16C, E38C, N59C, N83C,K94C, N104C and A124C.
 37. The full-length variant according to claim36, wherein said substitution is selected from the group consisting ofN16C, N59C and N16C+N59C.
 38. The full-length variant of claim 32,wherein said cysteine residue is covalently attached to a polymermolecule.
 39. The full-length variant according to claim 38, whereinsaid polymer molecule is a linear or branched polyethylene glycol. 40.The full-length variant according to claim 22, wherein said up to 10residue modifications comprises at least one introduced N-glycosylationsite and at least one introduced cysteine residue.
 41. (canceled) 42.The full-length variant of claim 1, wherein said variant comprises anamino acid sequence from residue no. 1 to residue no 131, which isidentical to the amino acid sequence from residue no. 1 to residue no.131 of huIFNG of SEQ ID NO:1.
 43. The full-length variant according toclaim 42, wherein said variant is un-glycosylated.
 44. The full-lengthvariant of claim 32, wherein said variant is glycosylated.
 45. Anucleotide sequence encoding the full-length variant of claim
 1. 46. Anexpression vector comprising a nucleotide sequence as defined in claim45.
 47. A host cell comprising a nucleotide sequence as defined in claim45 or an expression vector according to claim
 46. 48-49. (canceled) 50.A composition comprising a a full-length IFNG variant of claim 1 and acarrier.
 51. A pharmaceutical composition comprising the a full-lengthvariant of claim 1 and a pharmaceutically acceptable diluent, carrier oradjuvant. 52-55. (canceled)
 56. A method for treating or preventinginterstitial pulmonary diseases, said method comprising administering toa mammal, in particular a human being, in need thereof an effectiveamount of a full-length variant of claim
 1. 57-58. (canceled)
 59. Amethod for producing a full-length IFNG polypeptide, said methodcomprising i) cultivating a host cell as defined in claim 47 underconditions suitable for production of the IFNG polypeptide, and (ii)recovering the IFNG polypeptide.